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A Novel Rab5 GDP/GTP Exchange Factor Complexed to Rabaptin-5 Links Nucleotide Exchange to Effector Recruitment and Function
Abstract
The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.
Supplementary resource (1)
... Furthermore, multiple subunits of the Furthermore, we were able to identify three known Rab5 GEFs, of which two (GAPVD1 and ALS2/Alsin) are predominantly present in the GDP-locked form (Hunker et al., 2006;Topp et al., 2004). The third known and identified Rab5 GEF, RABGEF1/Rabex-5, was found in similar levels in both mutants, which can be explained by its ability to form a complex with the Rab5 effector Rabaptin-5 (Horiuchi et al., 1997;Z. Zhang et al., 2014). ...
... Finally, RABEP1 and RABGEF1 are known to interact and form a protein complex, yet they do not seem to follow the same binding patterns to the Rab11 paralogues; Rab11A appears to interact with both RABEP1 and RABGEF1 at similar strengths, whereas Rab11B appears to interact only with RABEP1 ( Fig. 3.3A). This suggests separate and additional functions for both proteins, besides the known Rab4-Rab5 activation bridge (Horiuchi et al., 1997;Kälin et al., 2015). ...
... Focusing on the Rab GTPases discussed above for their GTP-locked forms, GDP-locked Rab2A showed specific enrichment of the autophagy regulator CLEC16A, which has previously been reported with the C. elegans homologue (GOP-1) activating Rab2A homologue UNC-108 ( Fig. 3.8A) (Tam et al., 2017;Yin et al., 2017). The previously mentioned Rab5A GEFs (Alsin/ALS2, GAPVD1, and the RABEP1-RABGEF1 complex) are all high scoring hits for the GDP-form of Rab5A (Fig. 3.8B) (Horiuchi et al., 1997;Hunker et al., 2006;Topp et al., 2004;Z. Zhang et al., 2014). ...
Secretion and endocytosis are essential processes in eukaryotic cells, executed and tightly regulated by the cell’s endomembrane system. It is widely accepted that small GTPases of the Ras superfamily are major regulators of membrane trafficking and signalling, ensuring specificity and efficiency through their spatiotemporal regulation.Identifying the proteins interacting with small GTPases is vital for understanding how the small G proteins are regulated and where they function. With the aim of eliminating certain limitations posed by many traditional methods of studying protein-protein interactions, an adaptation of the recently developed in vivo proximity labelling technique BioID was used in this study. Here, BioID was redirected to the ectopic location of the surface of mitochondria (hereafter called MitoID) with the aim of restricting the non-specific background. Applying this method to 25 small GTPases of the Ras superfamily in their active and inactive states allowed for a direct and clean comparison between the putative interactomes, identifying both known effectors and regulators as well as putative novel interactors for most tested GTPases. Several potential novel interactions were validated through GST affinity chromatography and/or microscopy, namely with the Rab GTPases Rab2A (ARFGEF3/BIG3, STAMBPL1), Rab5A (OSBPL9, TBCK), Rab9A (HPS3, NDE1), and Rab11A/B (ALS2). Furthermore, numerous novel interactors were identified for Rab1A and Rab1B, two mammalian paralogues of the yeast protein Ypt1 that are known to be key regulators in ER to Golgi trafficking and suggested to play a role in the regulation of autophagy. The newly identified proteins include Rabaptin5, which is a key player in the regulation of endosomal trafficking, PPP1R37, a protein of unknown function, CALCOCO1 and CLEC16A, which are potentially involved in autophagy, and several components of the phosphatidylinsositol-3-phospate kinase (PI3K) complex I, which is known to be a major autophagic regulator. In collaboration with the Williams group, the PI3K complex I was shown to be a bona fide Rab1A regulator. Furthermore, direct binding assays showed that CALCOCO1, CLEC16A, and PPP1R37 all bind to Rab1A and Rab1B independently of adaptor proteins or other factors. More specifically, the Rab1 binding site on PPP1R37 was shown to be located on the C-terminal and central regions that contain multiple leucine rich repeats. Collectively, this study has shown that MitoID is an effective and powerful tool to study protein-protein interactions, and has identified and confirmed several novel interactors of small GTPases which can aid in gaining understanding of how major processes such as vesicle trafficking and autophagy are regulated.
... We examined the expression of all Rabs and related effector proteins in transcriptome datasets of developing mouse retina [29,30] from embryonic day 11 (E11) to postnatal day 28 (P28) and identified Rabgef1 as the only Rab-associated gene that showed increasing expression from P6 onwards (S1 Table). RabGEF1 functions during endocytosis as a GEF of Rab5 through formation of an obligatory and tight physical complex with Rabaptin-5 [31][32][33] (Fig 1A). When activated by the RabGEF1-Rabaptin5 complex, Rab5-GTP interacts with a variety of effector proteins including tethering factors such as Early Endosomal Antigen 1 (EEA1) and SNARE proteins such as Syntaxin13, leading to heterotypic fusion of clathrin-coated vesicles with early endosomes or homotypic fusion of early endosomes with each other [14,34] (Fig 1A). ...
... We then performed mass spectrometry analysis of the anti-RabGEF1 and IgG-immunoprecipitated proteins from control and KO retinal lysates. Our analysis confirmed an interaction between RabGEF1 and Rabaptin-5 (also known as Rabep1) in a 2:1 stoichiometry (Fig 5B), similar to previously published data [32,33]. We also observed an interaction of RabGEF1 with the Rabaptin-5 paralog Rabep2. ...
... In addition to promoting Rab5 activity as a GEF, RabGEF1 also binds ubiquitin [42] and its recruitment to the early endosome is controlled by ubiquitination [43]. Notably, the function of RabGEF1 has been investigated primarily in cell culture systems [32,42,44]. Only a few studies have been performed in vivo; specifically, the absence of RabGEF1 in mast cells and epidermal keratinocytes was demonstrated to contribute to skin inflammation in Rabgef1-KO mice [37,45]. ...
Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice ( Rabgef1 -/- ) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1 -/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1 -/- retina revealed altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype.
... In the next experiments, we examined whether inclusion of GTP-Rab5 that is known to be required for the fusion of early endosomes affects targeting of EE-SNARE liposomes. To this end, we purified prenylated versions of an active (GTPase-deficient) mutant (Rab5(Q79L)) and wt-Rab5 (converted into GDP-binding form in vitro) (see reference 35,36 , Supplementary Fig. S3a) and incorporated them into liposomes (Supplementary Fig. S2b and Supplementary Fig. S3b). The concentration of Rab5 protein in liposomes was comparable to that of endogenous endosomes (calibrated by comparing the intensities of Rab5 immunofluorescence signals of injected liposomes with those of endogenous endosomes, Supplementary Fig. S3c, d). ...
... All proteins were further purified by ion-exchange chromatography. Prenylated-Rab5 (WT and Q79L mutant) and prenylated-Rab7 (Q67L mutant) were expressed in Sf9 cells using a baculovirus system and purified by Mono-Q ion exchange column (GE Healthcare) 35 . All proteins were 95% pure, as judged by SDS-PAGE and Coomassie blue staining. ...
In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.
... Activator-inhibitor mechanisms for patterning typically presuppose the existence of both positive and negative feedback in Rho GTPase regulation, a supposition that is fulfilled by the observation that Rho GTPases can both positively and negatively regulate their own GEFs and GAPs [62][63][64][65] . Activation of Rho GTPase (that is, exchange of GDP for GTP) results from interaction with a GEF (guanine nucleotide exchange factor). ...
... The feedback mechanisms can be grouped based on the number of steps between the GTPase and the GEF or GAP (Fig. 3). First is direct feedback, where the active GTPase itself binds to a GEF and modifies its activity [62][63][64][65] . For example, at least seven Rho GEFs 66-68 and at least one CDC42 GEF 69 interact allosterically with RHO-GTP or CDC42-GTP, respectively, an interaction which directs the GEF to the plasma membrane and increases its activity. ...
The Rho GTPases — RHOA, RAC1 and CDC42 — are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function.
... A well-known example of a GEF-Rab-effector complex is Rabex5/Rab5/Rabaptin5 [62]. ...
... Rabex5 has GEF activity for Rab5 upon initial recruitment of Rab5 to the membrane [62]. ...
Cytokinesis is the final stage of cell division. Successful cytokinesis requires membrane trafficking pathways regulated by Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). Late in cytokinesis, an intercellular cytokinetic bridge (ICB) connecting the two daughter cells undergoes abscission, which requires depolymerization of actin. Rab35 recruits MICAL1 to oxidate and depolymerize actin filaments. We report that DENND2B, a protein previously implicated in cancer, mental retardation and multiple congenital disorders functions as a GEF for Rab35 and recruits and activates the GTPase at the ICB. Unexpectedly, the N terminal region of DENND2B interacts with an active mutant of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission resulting in increased multinucleated cells and over-accumulation of F-actin at the ICB. F-actin accumulation leads to formation of a chromatin bridge, a process known to activate the NoCut/abscission checkpoint, and DENND2B knockdown actives Aurora B kinase, a hallmark of checkpoint activation. This study identifies DENND2B as a crucial player in cytokinetic abscission and provides insight into the multisystem disorder associated with DENND2B mutation.
... Still, several other proteins involved in trafficking of endosomes and autophagosomes are linked to PD, including RabGEF1 and Arl8b (Bandres-Ciga et al., 2019). RabGEF1 activates Rab5 (Horiuchi et al., 1997) which characterizes early endosomes and is subsequently exchanged for Rab7. Both Rab7 and Arl8 are localized on late endosomes (Horiuchi et al., 1997;Jongsma et al., 2020;Marwaha et al., 2017), and Rab7 is exchanged for Arl8 during endosome maturation . ...
... RabGEF1 activates Rab5 (Horiuchi et al., 1997) which characterizes early endosomes and is subsequently exchanged for Rab7. Both Rab7 and Arl8 are localized on late endosomes (Horiuchi et al., 1997;Jongsma et al., 2020;Marwaha et al., 2017), and Rab7 is exchanged for Arl8 during endosome maturation . Thus, the autophago-lysosomal pathway is clearly important during PD pathogenesis, and stimulating autophagosome maturation can be a viable therapeutic strategy even without a causative role of Rab7 during PD pathogenesis. ...
During the pathogenesis of Parkinson's disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo. The disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or the T22N mutant as negative control. The cellular integrity was quantified by morphological and biochemical analyses. In primary neurons, WT Rab7 rescued the αSyn-induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn-induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain. In brief, Rab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.
... 59 Rabaptin-5, on the other hand, forms a complex with Rabex-5, a Rab5-GEF, and Rab5, thereby promoting Rab5 activation during endocytosis. 61,62 Because Rab5 activity is a key mediator of AMPAR endocytosis and LTD formation, 63 we explored whether phosphorylation of Rabaptin-5 at the PKD target site serine 407 is required to promote synaptic removal of AMPARs upon NMDA-induced LTD. ...
... Rabaptin-5 and the Rab5-GEF Rabex-5 function together to activate Rab5 and to promote the fusion of endocytic vesicles with early endosomes. 61,62,72 The small GTPase Rab5 plays a critical role in the regulation of constitutive and activity-dependent AMPAR endocytosis 63,73 and is essential for LTD. 63 By investigating another potential downstream target of PKD, the Rab5-GEF RIN1, we recently showed that enhancing Rab5 activity is a critical step during activity-dependent AMPAR internalization. ...
AMPA type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the Protein Kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their co-localization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.
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... In addition, several other proteins involved in tra cking of endosomes and autophagosomes are linked to PD, including RabGEF1 and Arl8b [24]. RabGEF1 activates Rab5 [60] which characterizes early endosomes and is subsequently exchanged for Rab7. Both Rab7 and Arl8 are localized on late endosomes [60][61][62], and Rab7 is exchanged for Arl8 during endosome maturation [62]. ...
... RabGEF1 activates Rab5 [60] which characterizes early endosomes and is subsequently exchanged for Rab7. Both Rab7 and Arl8 are localized on late endosomes [60][61][62], and Rab7 is exchanged for Arl8 during endosome maturation [62]. Thus, the autophagolysosomal pathway clearly is important during PD pathogenesis, and stimulating autophagosome maturation can be a viable therapeutic strategy even without a causative role of Rab7 during PD pathogenesis. ...
Background
During the pathogenesis of Parkinson’s disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo .Methods
The disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or its dominant-negative T22N mutant as a control. The cellular integrity was quantified by morphological and biochemical analyses.ResultsIn primary neurons, WT Rab7 rescued the αSyn -induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn -induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain.Conclusion
Rab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.
... Rab5-vesicles then uncoat AP2 adaptor complexes and coat proteins, a process required for vesicle fusion with EEs (Semerdjieva et al., 2008). Lastly, Rab5 recruits various effectors, such as VPS34, EEA1, and Rabaptin-5/Rabex-5 complex (Stenmark et al., 1995;Horiuchi et al., 1997;Rubino et al., 2000), through which the endocytic vesicles dock and fuse with EE membrane. ...
... Stenmark et al. (1995),Horiuchi et al. (1997),McLauchlan et al. (1998),Rubino et al. (2000),Leonard et al. (2008),Satoh et al. (2008),Semerdjieva et al. (2008), von Zastrow and Williams(2012), Kanamori et al. (2015), Zhang et al. (2014), Wang et al. (2017), and Moya-Alvarado et al. al. (2000), Seachrist and Ferguson (2003), Odley et al. (2004), Zheng et al. (2008), Yudowski et al. (2009), Hoogenraad et al. (2010), Esseltine et al. (2011), D'Souza et al. (2014), andGromova et al. al. (1996),Sönnichsen et al. (2000),Park et al. (2004),Zhang et al. (2004),Park et al. (2006),Wang et al. (2008),Horgan et al. (2010),Kelly et al. (2012),Lazo et al. (2013),Kramer et al. (2019), andLin et al. (2020) ...
Due to their enormous surface area compared to other cell types, neurons face unique challenges in properly handling supply and retrieval of the plasma membrane (PM)-a process termed PM turnover-in their distal areas. Because of the length and extensiveness of dendritic branches in neurons, the transport of materials needed for PM turnover from soma to distal dendrites will be inefficient and quite burdensome for somatic organelles. To meet local demands, PM turnover in dendrites most likely requires local cellular machinery, such as dendritic endocytic and secretory systems, dysregulation of which may result in dendritic pathology observed in various neurodegenerative diseases (NDs). Supporting this notion, a growing body of literature provides evidence to suggest the pathogenic contribution of dysregulated PM turnover to dendritic pathology in certain NDs. In this article, we present our perspective view that impaired dendritic endocytic and secretory systems may contribute to dendritic pathology by encumbering PM turnover in NDs.
... Likewise, TBC-2 is required for the downregulation of RAB-5 activity during dense core vesicle sorting and releasing in neurons (Chotard et al., 2010b;Sasidharan et al., 2012). The C. elegans genome encodes three putative RAB-5-GEF proteins (Horiuchi et al., 1997), including RIN-1, RABX-5, and RME-6 (Doi et al., 2013;Poteryaev et al., 2010;Sato et al., 2005). Although the mammalian homolog of RIN-1, Rin1, functions as a GEF for Rab5, RIN-1 specifically interacts with the GTP-bound form of CED-10/Rac1 and directs actin remodeling (Doi et al., 2013). ...
... Being a Rab5 effector, Rabaptin-5 has been shown to form a complex with Rabex-5 and regulate its GEF activity (Horiuchi et al., 1997;Lippé et al., 2001). RABN-5 is the ortholog of mammalian Rabaptin-5 in C. elegans. ...
To explore the mechanism of Rab5/RAB-5 activation during endocytic recycling, we perform a genome-wide RNAi screen and identify a recycling regulator, LET-502/ROCK. LET-502 preferentially interacts with RAB-5(GDP) and activates RABX-5 GEF activity toward RAB-5, presumably by disrupting the self-inhibiting conformation of RABX-5. Furthermore, we find that the concomitant loss of LET-502 and another CED-10 effector, TBC-2/RAB-5-GAP, results in an endosomal buildup of RAB-5, indicating that CED-10 directs TBC-2-mediated RAB-5 inactivation and re-activates RAB-5 via LET-502 afterward. Then, we compare the functional position of LET-502 with that of RME-6/RAB-5-GEF. Loss of LET-502-RABX-5 module or RME-6 leads to diminished RAB-5 presence in spatially distinct endosome groups. We conclude that in the intestine of C. elegans, RAB-5 resides in discrete endosome subpopulations. Under the oversight of CED-10, LET-502 synergizes with RABX-5 to revitalize RAB-5 on a subset of endosomes in the deep cytosol, ensuring the progress of basolateral recycling.
... 62 Rabex5 has GEF activity for Rab5 upon initial recruitment of Rab5 to the membrane. 62 Once Rab5 is activated on the membrane, it interacts with its effector Rabaptin5. 63 Subsequently, Rabaptin5 binds to Rabex5 and increases its GEF activity, thus ensuring sustained GTPase activation and effector function. ...
Cytokinesis relies on membrane trafficking pathways regulated by Rabs and guanine nucleotide exchange factors (GEFs). During cytokinesis, the intercellular cytokinetic bridge (ICB) connecting daughter cells undergoes abscission, which requires actin depolymerization. Rab35 recruits MICAL1 to oxidize and depolymerize actin filaments. We show that DENND2B, a protein linked to cancer and congenital disorders, functions as a Rab35 GEF, recruiting and activating Rab35 at the ICB. DENND2B's N-terminal region also interacts with an active form of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission, leading to multinucleated cells and filamentous actin (F-actin) accumulation at the ICB, impairing recruitment of ESCRT-III at the abscission site. Additionally, F-actin accumulation triggers the formation of a chromatin bridge, activating the NoCut/abscission checkpoint, and DENND2B knockdown activates Aurora B kinase, a hallmark of checkpoint activation. Thus, our study identifies DENND2B as a crucial player in cytokinetic abscission.
... 63 Control of early efferosome formation is governed by Rab5-GTP, which possesses fusion activity due to the activation and stabilization of several effectors, such as early endosomal antigen 1 (EEA1) 64 and complex of Rabex-5 and Rabaptin-5. 65 The process at this stage is relatively slow, with a half-life of approximately 15 min. Subsequently, the efferosome matures continuously from early to late stages, which is accompanied by the loss of Rab5-GTP and the acquisition of Rab7-GTP, mediated by the class C homotypic fusion and vacuole protein sorting (HOPS) complex. ...
Increasing evidence indicates that chronic, low-grade inflammation is a significant contributor to the fundamental pathogenesis of type 2 diabetes mellitus (T2DM). Efferocytosis, an effective way to eliminate apoptotic cells (ACs), plays a critical role in inflammation resolution. Massive accumulation of ACs and the proliferation of persistent inflammation caused by defective efferocytosis have been proven to be closely associated with pancreatic islet β cell destruction, adipose tissue inflammation, skeletal muscle dysfunction, and liver metabolism abnormalities, which together are considered the most fundamental pathological mechanism underlying T2DM. Therefore, here we outline the association between the molecular mechanisms of efferocytosis in glucose homeostasis, T2DM, and its complications, and we analyzed the present constraints and potential future prospects for therapeutic targets in T2DM and its complications.
... an effector for Rab5, which will lead to further recruitment of Rabex5 to sites of Rab5 activation [55]. The system encodes for an ultrasensitive positive feedback loop [56,57]. ...
Rab GTPases are molecular switches with essential roles in mediating vesicular trafficking and establishing organelle identity. The conversion from the inactive, cytosolic to the membrane-bound, active species and back is tightly controlled by regulatory proteins. Recently, the roles of membrane properties and lipid composition of different target organelles in determining the activity state of Rabs have come to light. The investigation of several Rab guanine nucleotide exchange factors (GEFs) has revealed principles of how the recruitment via lipid interactions and the spatial confinement on the membrane surface contribute to spatiotemporal specificity in the Rab GTPase network. This paints an intricate picture of the control mechanisms in Rab activation and highlights the importance of the membrane lipid code in the organization of the endomembrane system.
... Rabex5 is the canonical Rab5GEF with clearly established roles in endosomal fusion and sorting into EEA1-positive endosomes (Horiuchi et al., 1997). Rin1 has a Ras binding domain and mediates Rabex5-dependent ubiquitination of endosomal Ras leading to its attenuation (Xu et al., 2010). ...
Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome.
Summary statement
Here we demonstrate how Rme-6, a Rab5GEF, co-ordinates trafficking and signalling of EGFR on the early endocytic pathway to ensure appropriate regulation of downstream ERK1/2 signalling.
... All these interactions contribute to a feed forward loop of Rab5 recruitment to membranes by bringing closer Rab5 to its GEF and to already activated and membrane bound Rab5, likely promoting biased binding of Rab5-GTP to membranes and promoting domain formation [3,14]. The interaction of Rabaptin5 with Rabex5 has been shown to produce structural changes on Rabex5 structure and potentiate its activity [9,11,12,[15][16][17]. Besides a possible first step of this domain formation may pass through Rabex5 recruitment to Early Endosomes (EE) via two distinct Ubiquitin binding domains [18]. ...
Cellular processes are regulated by the formation of specific membrane domains with different lipid and protein compositions. Small GTPases play a role in symmetry breaking and compartmentalization, with early endosomes presenting Rab5-enriched domains that regulate vesicle tethering and fusion. Rabaptin5, which binds activated Rab5 and forms dimers, is essential for endosome fusion and promotes Rab5 recruitment to membranes. Liquid-liquid phase separation (LLPS) is a mechanism for the biogenesis and maintenance of membrane-less organelles and intracellular organization. We show that several partners of Rab proteins, such as Rabaptin5, have phase-separation properties. In particular, Rabaptin5 form condensates close to membranes, while promoting the enrichment of several Rabs involved in early steps of endocytosis. We propose that phase separation of Rab partners ensures efficient recruitment of their respective Rabs and domain formation and maintenance.
... These two cascade mechanisms can work in a countercurrent manner to direct a programmed series of Rab transitions as membrane flows along a membrane traffic pathway. In addition, positivefeedback loops are formed through the ternary interactions of a Rab GEF, the activated Rab, and one of its effectors (Horiuchi et al., 1997;Medkova et al., 2006). These positive-feedback loops can function to sharpen Rab transitions and contribute to the maturation of carrier vesicles (Mizuno-Yamasaki et al., 2010;Stalder et al., 2013). ...
Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport. To explore this proposal we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab Ypt51. The Sec2GEF-GFP-CUE construct localized to bright puncta predominantly near sites of polarized growth and this localization was dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth despite the misdirection of Sec4 and its effectors. A low efficiency mechanism for localization of Sec2 to secretory vesicles that is independent of known cues might be responsible. In total, the results suggest that while Rabs may play a critical role in specifying the direction of vesicular transport cells are remarkably tolerant of Rab misdirection.
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... Previous studies have shown that Rab5 can exist as Rab5-GTP (membrane bound) and Rab5-GDP (cytosolic) forms in the cell. 7,26,27 We performed co-immunoprecipitation (coIP) analysis to assess the interaction between Rab5 and NM IIs. We transiently expressed GFPtagged wild-type (WT) Rab5, constitutively active Rab5-Q79L-GFP (GTP bound form, membranous), and dominant-negative (DN) Rab5-S34N-GFP (GDP bound form, cytosolic) in MDA-MB 231 cells, and immunoprecipitated the exogenous Rab5s with antibodies specific for GFP. ...
Endocytic vesicular trafficking requires merging of two lipid bilayers, but how the two lipid bilayers can come close together during fusion and fission in endocytic trafficking is not well explored. Here, we establish that knocking down nonmuscle myosin IIs (NM IIs) by small interfering RNA (siRNA) or inhibition of their activities by (-) blebbistatin causes the formation of a ring-like assembly of early endosomes (raEE). Inhibition of NM II assembly by an inhibitor of regulatory light-chain (RLC) kinase results in the formation of raEE, whereas inhibition of NM II disassembly by inhibitors of heavy chain kinases, protein kinase C (PKC) and casein kinase 2 (CK2), causes the dispersion of early endosomes. The raEEs retain EEA1, Rab7, and LAMP2 markers. Overexpression of an assembly incompetent form, RLC-AA, and disassembly incompetent form, NMHCIIB-S6A or NMHCIIA-1916A, induces such defects, respectively. Altogether, these data support that NM II assembly and disassembly dynamics participate in endocytic trafficking by regulating fission to maintain the size of early endosomes.
... These two cascade mechanisms can work in a counter-current fashion to direct a programmed series of Rab transitions as membrane flows along a membrane traffic pathway. In addition, positive feedback loops are formed through the ternary interactions of a Rab GEF, the activated Rab and one of its effectors (Horiuchi et al., 1997;Medkova et al., 2006). These positive feedback loops can function to sharpen Rab transitions and contribute to the maturation of carrier vesicles (Mizuno-Yamasaki et al., 2010;Stalder et al., 2013). ...
Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport because anterograde vesicles are marked with a different Rab than retrograde vesicles. To explore this proposal we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab Ypt51. The Sec2GEF-GFP-CUE construct was found to localize to bright puncta predominantly near sites of polarized growth and this localization was dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. Nonetheless the Sec2GEF-GFP-CUE puncta were largely static and did not appear to fuse with the plasma membrane, suggesting that recruitment of an exocytic Rab is not sufficient to reverse the direction of an endocytic compartment. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth, where they tethered and fused with the plasma membrane despite the misdirection of Sec4 and its effectors. A low efficiency mechanism for localization of Sec2 to secretory vesicles that is independent of known cues might be responsible. In total, the results suggest that while Rabs may play a critical role in specifying the direction of vesicular transport they cannot act alone in this regard.
... Two Rab family members, early endosomal Rab5 (Vps21, Ypt52, Ypt53, and Ypt10 in yeasts) and late endosomal Rab7 (Ypt7 in yeasts) are key Rabs that coordinate endosome maturation (Huotari and Helenius, 2011;Langemeyer et al., 2020;Borchers et al., 2021). After endocytosis, Rab5 is activated by a specific GEF, such as Rabex-5 (Horiuchi et al., 1997), and it facilitates the formation of early endosomes and their homotypic fusion (Stenmark et al., 1994). The early endosomal Rab5 then directly recruits the Mon1-Ccz1 heterodimeric complex, a Rab7-GEF, to the same compartment (Nordmann et al., 2010;Kinchen and Ravichandran, 2010). ...
Rab5 and Rab7 are known to regulate endosome maturation, and a Rab5-to-Rab7 conversion mediated by a Rab7 activator, Mon1–Ccz1, is essential for progression of the maturation process. However, the importance and mechanism of Rab5 inactivation during endosome maturation are poorly understood. Here, we report a novel Rab5-GAP, TBC1D18, which is associated with Mon1 and mediates endosome maturation. We found that increased active Rab5 (Rab5 hyperactivation) in addition to reduced active Rab7 (Rab7 inactivation) occurs in the absence of Mon1. We present evidence showing that the severe defects in endosome maturation in Mon1-KO cells are attributable to Rab5 hyperactivation rather than to Rab7 inactivation. We then identified TBC1D18 as a Rab5-GAP by comprehensive screening of TBC-domain-containing Rab-GAPs. Expression of TBC1D18 in Mon1-KO cells rescued the defects in endosome maturation, whereas its depletion attenuated endosome formation and degradation of endocytosed cargos. Moreover, TBC1D18 was found to be associated with Mon1, and it localized in close proximity to lysosomes in a Mon1-dependent manner.
... Small GTPase proteins are known to play an important role in numerous cellular processes, including cell proliferation and growth (Militello and Colombo, 2013;Park and Bi, 2007), cytoskeletal regulation and cell motility (Charest and Firtel, 2007) and intracellular trafficking (Murphy et al., 1996;Ridley, 2006;Zerial and McBride, 2001). Some of these processes are hypothesized to depend on the emergence of spatial patterns formed as a result of nonlinear interactions of small GTPases with their effectors on membranes (Horiuchi et al., 1997;Zerial and McBride, 2001) (see Supplementary Section S1 for details). Although the key molecular players and the interactions between them have been discovered a while ago and have been included in a mechanistic model of pattern formation for small GTPases (Goryachev and Pokhilko, 2008), the recent reconstitution of domain formation of the small GTPase Rab5 by the GDP dissociation inhibitor (GDI) and the guanine nucleotide exchange factor (GEF)/effector Rabex5/Rabaptin5 complex by Cezanne et al. (2020) motivates alternative models for the small GTPase Rab5. ...
Motivation:
Access to unprecedented amounts of quantitative biological data allows us to build and test biochemically accurate reaction-diffusion models of intracellular processes. However, any increase in model complexity increases the number of unknown parameters and, thus, the computational cost of model analysis. To efficiently characterize the behavior and robustness of models with many unknown parameters remains, therefore, a key challenge in systems biology.
Results:
We propose a novel computational framework for efficient high-dimensional parameter space characterization of reaction-diffusion models in systems biology. The method leverages the Lp-Adaptation algorithm, an adaptive-proposal statistical method for approximate design centering and robustness estimation. Our approach is based on an oracle function, which predicts for any given point in parameter space whether the model fulfills given specifications. We propose specific oracles to efficiently predict four characteristics of Turing-type reaction-diffusion models: bistability, instability, capability of spontaneous pattern formation and capability of pattern maintenance. We benchmark the method and demonstrate that it enables global exploration of a model's ability to undergo pattern-forming instabilities and to quantify robustness for model selection in polynomial time with dimensionality. We present an application of the framework to pattern formation on the endosomal membrane by the small GTPase Rab5 and its effectors, and we propose molecular mechanisms underlying this system.
Availability and implementation:
Our code is implemented in MATLAB and is available as open source under https://git.mpi-cbg.de/mosaic/software/black-box-optimization/rd-parameter-space-screening.
Supplementary information:
Supplementary data are available at Bioinformatics online.
... Studies in various systems have revealed multiple Rab5 GEF proteins, including RABX-5/Rabex-5 [109,150,149,153], hRME-6/RME-6 [112], and Rin2/RIN-1 [29]. RABX-5/Rabex-5 is the first protein identified as a small GTPase GEF and is primarily localized at early endosomes [52]. During degradation transport from early to late endosomes, Table 1 Rab proteins and their effectors in endocytic transport. ...
After endocytosis, diverse cargos are sorted into endosomes and directed to various destinations, including extracellular macromolecules, membrane lipids, and membrane proteins. Some cargos are returned to the plasma membrane via endocytic recycling. In contrast, others are delivered to the Golgi apparatus through the retrograde pathway, while the rest are transported to late endosomes and eventually to lysosomes for degradation. Rab GTPases are major regulators that ensure cargos are delivered to their proper destinations. Rabs are localized to distinct endosomes and play predominant roles in membrane budding, vesicle formation and motility, vesicle tethering, and vesicle fusion by recruiting effectors. The cascades between Rabs via shared effectors or the recruitment of Rab activators provide an additional layer of spatiotemporal regulation of endocytic trafficking. Notably, several recent studies have indicated that disorders of Rab-mediated endocytic transports are closely associated with diseases such as immunodeficiency, cancer, and neurological disorders.
... For clathrinmediated endocytosis, Rab GTPases are crucial regulators of endosome maturation [15][16][17] . Early endosomes accumulate active Rab5 via a positive feedback loop consisting of the Rab5-specific guanine nucleotide exchange factor (GEF) Rabex5 and effector protein Rabaptin5 18,19 . As early endosomes convert into late endosomes, Rab5 together with PI3P recruits a protein complex containing Mon1 and Ccz1 20,21 . ...
Macropinocytosis, an evolutionarily conserved mechanism mediating nonspecific bulk uptake of extracellular fluid, has been ascribed diverse functions. How nascent macropinosomes mature after internalization remains largely unknown. By searching for proteins that localize on macropinosomes during the Rab5-to-Rab7 transition stage in Dictyostelium, we uncover a complex composed of two proteins, which we name PripA and TbcrA. We show that the Rab5-to-Rab7 conversion involves fusion of Rab5-marked early macropinosomes with Rab7-marked late macropinosomes. PripA links the two membrane compartments by interacting with PI(3,4)P2 and Rab7. In addition, PripA recruits TbcrA, which acts as a GAP, to turn off Rab5. Thus, the conversion to Rab7 is linked to inactivation of the upstream Rab5. Consistently, disruption of either pripA or tbcrA impairs Rab5 inactivation and macropinocytic cargo processing. Therefore, the PripA-TbcrA complex is the central component of a Rab GAP cascade that facilitates programmed Rab switch and efficient cargo trafficking during macropinosome maturation. Macropinocytosis has emerged as an evolutionarily conserved mechanism for bulk uptake of extracellular fluid. Here they show how a Rab GAP cascade centered on the PripA-TbcrA complex facilitates Rab conversion and macropinosome maturation.
... The conversion of GDP-bound to GTP-bound form results in the activation of Rab5 that promotes endocytosis and endocytic traffic (Bucci et al., 1992;Xu et al., 2016). Thus, Rabex5 was the first guanine nucleotide exchange factor (GEF) identified for the small GTPase Rab5 (Horiuchi et al., 1997). A detailed structural analysis of Rabex5 showed that it contained a Vps9 catalytic domain that was responsible for its GEF activity towards Rab5 (Hama et al., 1999). ...
Ras and Rab interactor 3 (RIN3) functions as a Guanine nucleotide Exchange Factor (GEF) for some members of the Rab family of small GTPase. By promoting the activation of Rab5, RIN3 plays an important role in regulating endocytosis and endocytic trafficking. In addition, RIN3 activates Ras, another small GTPase, that controls multiple signaling pathways to regulate cellular function. Increasing evidence suggests that dysregulation of RIN3 activity may contribute to the pathogenesis of several disease conditions ranging from Paget’s Disease of the Bone (PDB), Alzheimer’s Disease (AD), Chronic Obstructive Pulmonary Disease (COPD) and to obesity. Recent genome-wide association studies (GWAS) identified variants in the RIN3 gene to be linked with these disease conditions. Interestingly, some variants appear to be missense mutations in the functional domains of the RIN3 protein while most variants are located in the noncoding regions of the RIN3 gene, potentially altering its gene expression. However, neither the protein structure of RIN3 nor its exact function(s) (except for its GEF activity) has been fully defined. Furthermore, how the polymorphisms/variants contribute to disease pathogenesis remain to be understood. Herein, we examine, and review published studies in an attempt to provide a better understanding of the physiological function of RIN3; More importantly, we construct a framework linking the polymorphisms/variants of RIN3 to altered cell signaling and endocytic traffic, and to potential disease mechanism(s).
... This excellent study led to the identification of the vacuolar H+ ATPase pump (V-ATPase), whose main role consists in lysosome acidification. Following this period, from the 1980s until the early 2000s, extraordinary works were accomplished related to identifying the machinery of membrane traffic on the biosynthetic and endocytic routes to lysosomes (Kielian et al., 1986;Chavrier et al., 1990;Schu et al., 1993;Ullrich et al., 1994;Méresse et al., 1995;Horiuchi et al., 1997;Christoforidis et al., 1999;Lewis et al., 2000). Clear examples amongst many are the characterization of the vacuolar protein sorting (VPS) genes in yeast, mammalian orthologs of which were subsequently shown to make up much of the core machinery for delivery of cargo to and from lysosomes, the identification of the ESCRT proteins, crucial for multivesicular body (MVB) formation and the identification of the small Rab GTPases and their regulators and effectors necessary for the endocytic pathway to lysosomes (Katzmann et al., 2001). ...
For a long time, lysosomes were considered as mere waste bags for cellular constituents. Thankfully, studies carried out in the past 15 years were brimming with elegant and crucial breakthroughs in lysosome research, uncovering their complex roles as nutrient sensors and characterizing them as crucial multifaceted signaling organelles. This review presents the scientific knowledge on lysosome physiology and functions, starting with their discovery and reviewing up to date ground-breaking discoveries highlighting their heterogeneous functions as well as pending questions that remain to be answered. We also review the roles of lysosomes in anti-cancer drug resistance and how they undergo a series of molecular and functional changes during malignant transformation which lead to tumor aggression, angiogenesis, and metastases. Finally, we discuss the strategy of targeting lysosomes in cancer which could lead to the development of new and effective targeted therapies.
... Two Rab family members, early endosomal Rab5 (Ypt5 in yeasts) and late endosomal Rab7 (Ypt7 in yeasts) are key Rabs that coordinate endosome maturation (Huotari & Helenius, 2011;Borchers et al., 2021). After endocytosis, Rab5 is activated by a specific GEF, such as Rabex5 (Horiuchi et al., 1997), and it facilitates the formation of early endosomes and their homotypic fusion (Stenmark et al., 1994). The early endosomal Rab5 then directly recruits the Mon1-Ccz1 heterodimeric complex, a Rab7-GEF, to the same compartment (Nordmann et al., 2010;Kinchen & Ravichandran, 2011), and the recruited Mon1-Ccz1 activates Rab7, which regulates subsequent late endosome maturation. ...
Endosome maturation is essential for efficient degradation of internalized extracellular molecules and plasma membrane proteins. Two Rab GTPases, Rab5 and Rab7, are known to regulate endosome maturation, and a Rab5-to-Rab7 conversion mediated by a Rab7 activator, Mon1-Ccz1, is essential for progression of the maturation process. However, the importance and mechanism of Rab5 inactivation during endosome maturation is poorly understood. Here we report a novel Rab5 inactivator (Rab5-GTPase activating protein [Rab5-GAP]), TBC1D18, which is associated with Mon1 and mediates endosome maturation. We found that Rab5 hyperactivation in addition to Rab7 inactivation occurs in the absence of Mon1. We present evidence showing that the severe defects in endosome maturation observed in Mon1-KO cells are attributable to Rab5 hyperactivation rather than to Rab7 inactivation. We then identified TBC1D18 as a Rab5-GAP by comprehensive screening of TBC-domain-containing Rab-GAPs. Expression of TBC1D18 in Mon1-KO cells rescued the defects in endosome maturation, whereas its depletion attenuated endosome formation and degradation of endocytosed cargos. Moreover, TBC1D18 was found to be able to interact with Mon1, and it localized in close proximity to lysosomes in a Mon1-dependent manner. Thus, TBC1D18 is a crucial regulator of endosome maturation that functions together with Mon1.
... Previously, Rabaptin5 had been characterized as a regulator of Rab5 activity by complex formation with Rabex5, the GDP/GTP exchange factor of Rab5. In addition, it had been shown to bind to Rab4ÁGTP and Rab5ÁGTP, the hallmark Rab proteins of early endosomes (Horiuchi et al, 1997;Lipp e et al, 2001;Mattera et al, 2006;K€ alin et al, 2015. Rabaptin5 thus was known to contribute to endosome identity and maturation (Huotari & Helenius, 2011). ...
Selective autophagy of damaged organelles is important to maintain cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here, we identify two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Autophagy of early endosomes damaged by chloroquine or monensin treatment requires Rabaptin5 and particularly a short sequence motif that binds to the WD domain of ATG16L1. Rabaptin5 and its interaction with ATG16L1 further contributes to the autophagic elimination of Salmonella enterica early after infection, when it resides in phagosomes with early endosomal characteristics. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective targeting of autophagy to damaged early endosomes and phagosomes.
... Autoinhibition controls the Rab5 GEF Another widely used regulatory mechanism is the autoinhibition of GEFs to control their activity. This has been analyzed in detail for the early endosomal Rab5-specific GEF Rabex-5, which interacts with the Rab5-effector Rabaptin-5 (Horiuchi et al., 1997). One factor for Rabex-5 recruitment to endocytic vesicles are ubiquitinated cargo proteins at the plasma membrane (Fig. 3 D ;Mattera et al., 2006;Lee et al., 2006). ...
The eukaryotic endomembrane system consists of multiple interconnected organelles. Rab GTPases are organelle-specific markers that give identity to these membranes by recruiting transport and trafficking proteins. During transport processes or along organelle maturation, one Rab is replaced by another, a process termed Rab cascade, which requires at its center a Rab-specific guanine nucleotide exchange factor (GEF). The endolysosomal system serves here as a prime example for a Rab cascade. Along with endosomal maturation, the endosomal Rab5 recruits and activates the Rab7-specific GEF Mon1-Ccz1, resulting in Rab7 activation on endosomes and subsequent fusion of endosomes with lysosomes. In this review, we focus on the current idea of Mon1-Ccz1 recruitment and activation in the endolysosomal and autophagic pathway. We compare identified principles to other GTPase cascades on endomembranes, highlight the importance of regulation, and evaluate in this context the strength and relevance of recent developments in in vitro analyses to understand the underlying foundation of organelle biogenesis and maturation.
... While isolation of homologous cDNAs like Rab7 from Vigna aconitifolia is now also possible by low-stringency screening method using heterologous probes (Feng et al., 2014) and rgp2 from rice . Identified Rab5 homolog clone and sequenced 130 randomly selected clones from a maize leaf cDNA library (Horiuchi et al., 1997). Rhal protein has been found to be localized to early endosomes and highly homologous to Rab5. ...
The mutations are genetic changes in the genome sequences and have a significant role in biotechnology, genetics, and molecular biology even to find out the genome sequences of a cell DNA along with the viral RNA sequencing. The mutations are the alterations in DNA that may be natural or spontaneous and induced due to biochemical reactions or radiations which damage cell DNA. There is another cause of mutations which is known as transposons or jumping genes which can change their position in the genome during meiosis or DNA replication. The transposable elements can induce by self in the genome due to cellular and molecular mechanisms including hypermutation which caused the localization of transposable elements to move within the genome. The use of induced mutations for studying the mutagenesis in crop plants is very common as well as a promising method for screening crop plants with new and enhanced traits for the improvement of yield and production. The utilization of insertional mutations through transposons or jumping genes usually generates stable mutant alleles which are mostly tagged for the presence or absence of jumping genes or transposable elements. The transposable elements may be used for the identification of mutated genes in crop plants and even for the stable insertion of transposable elements in mutated crop plants. The guanine nucleotide-binding (GTP) proteins have an important role in inducing tolerance in rice plants to combat abiotic stress conditions.
... A vast network of interaction partners of Rab5 has been identified, providing Rab5 with one of the most complex interactomes among the Rab family (Christoforidis et al., 1999). This includes GEFs like Rabex-5 and RIN1 (Horiuchi et al., 1997;Tall et al., 2001), but also Rab5specific GAPs such as RN-Tre (Lanzetti et al., 2000) and Rab-GAP5 (Haas et al., 2005). Within the large pool of downstream effectors, early endosome antigen 1 (EEA1) is probably the best RNA transport serves as a prime example of how spatio-temporal control can influence the expression of genes, underlying essential biological processes such as embryonic development or neuronal plasticity (Medioni et al., 2012;Mofatteh, 2020). ...
Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction is linked to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and the early endosome in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, in which two arm-like appendages, each consisting of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. We demonstrate that the coiled-coil domains of Fy-2 are flexible and project into opposite directions from the FERRY complex core. While the C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils together with Fy-5 bind mRNA. Thus, Fy-2 serves as binding hub that connects not only all five complex subunits, but also mediates the binding to mRNA and to the early endosome via Rab5. The FERRY structure provides novel mechanistic insight into long-distance mRNA transport.
... membranes du compartiment, passant de Rab5 à Rab7 en seulement quelques minutes (Figure 18;Rink et al., 2005;Vonderheit et Helenius, 2005). Rab5 est maintenue active sur les membranes grâce à sa GEF Rabex-5(Horiuchi et al., 1997), elle-même positivement régulée par son interaction avec Rabaptin-5, effecteur de Rab5(Christoforidis, McBride, et al., 1999;Lippe et al., 2001).Il en résulte une boucle d'activation positive recrutant et maintenant Rab5 sur la membrane des endosomes précoces, et dont le maintien est directement dépendant de Rabex-5. Ce sont les protéines SAND-1/Mon1a/b qui interviennent pour mettre fin à cette boucle et permettre la conversion. ...
Plus d’une soixantaine de protéines différentes sont insérées dans la membrane du lysosome et participent aux différentes fonctions de cet organite. Mais les voies de transport intracellulaire qui mènent les protéines membranaires lysosomales (LMP) jusqu’à cet organite ne sont pas bien comprises. De façon intéressante, certaines LMP présentent des localisations intracellulaires anormales dans certains cancers. C’est notamment le cas de la glycoprotéine LAMP1 qui s’avère surexposée à la surface cellulaire dans plusieurs cancers, où elle y joue plusieurs rôles dans l’agressivité tumorale. Grâce au système RUSH (pour Retention Using Selective Hooks), permettant la synchronisation du transport le long de la voie de sécrétion, nous avons montré que LAMP1, après néosynthèse, passe par la membrane plasmique avant d’accéder aux endosomes. La comparaison des voies empruntées par différentes LMP a aussi permis de révéler que LAMP1 et LIMP2 sont triées au niveau de l’appareil de Golgi, LIMP2 étant concentrée dans des structures vésiculaires caractéristiques et dépourvues de clathrine. Nous avons aussi montré que, de façon surprenante, ce tri au niveau de l’appareil de Golgi est indépendant des signaux de recrutement d’adaptateurs à la clathrine portés dans les queues C-terminales de ces deux LMP. Afin d’étudier quels mécanismes pourraient être impliqués dans la surexposition de LAMP1 à la surface cellulaire de certains cancers, nous avons aussi réalisé un criblage d’inactivation de gènes basé sur la technologie CRISPR-Cas9. Nous avons sélectionné les gènes dont l’inactivation affectait les niveaux de LAMP1 en surface et nous avons obtenu de nombreux gènes candidats qui sont à l’étude.
... At these junction structures, Rtn3L could coordinate endosome maturation by regulating motor loading and dissociation, potentially together with other ER-endosome MCS proteins that regulate similar processes including protrudin, RILP, and ORP1L (Johansson et al., 2007;Raiborg et al., 2015;Rocha et al., 2009). Second, an assortment of factors can regulate endosome maturation including Rab guanine nucleotide exchange factors, Rab GTPase-activating proteins, guanine nucleotide dissociation in-hibitor (GDI), GDI-displacement factor, and endosome homotypic fusion machineries (Gougeon et al., 2002;Horiuchi et al., 1997;Hutt et al., 2000;Kinchen and Ravichandran, 2010;Nickerson et al., 2009;Poteryaev et al., 2007Poteryaev et al., , 2010Sivars et al., 2003;Wang et al., 2003). Rtn3L-mediated MCSs could provide a platform for the recruitment of these factors. ...
ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.
... We determined increased levels of Rab5 activation in 6-month-old PA-Rab5 mouse synaptosomes (SSs), compared with WT, by both IP with the Rab5-GTP specific antibody and by GTP-agarose pull-down of Rab5-GTP (Figures 2G and S2G). Also, the synaptosomal levels of the Rab5 effector Rab-GEF-1 (Horiuchi et al., 1997) and those of the GTPase-activating protein RabGAP5 were increased and decreased, respectively, in PA-Rab5, compared to WT, reflecting Rab5 overactivation (Figure 2H). Further extraction of total SS into SS soluble/cytosolic (SScyt) and post-synaptic density (PSD)/membrane fractions (Figures 2I and S2H) revealed a significant increase in the ratio of soluble:membrane (SScyt:PSD), or conversely, a significant decrease in the ratio of membrane:soluble (PSD:SScyt), for p-GluA1 S831 and p-GluA1 S845 in PA-Rab5 ( Figures 2I and 2J), compared to WT mice, consistent with AMPAR removal from the PSD membrane and internalization by endocytosis (Brown et al., 2005). ...
Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer’s disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-β secretase cleaved C-terminal fragment (APP-βCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-βCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyperactivation of Rab5 comparable to that in AD brain reproduces AD-related Rab5-endosomal enlargement and mistrafficking, hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss, and tau hyperphosphorylation via activated glycogen synthase kinase-3β. Importantly, Rab5-mediated endosomal dysfunction induces progressive cholinergic neurodegeneration and impairs hippocampal-dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a pathogenic cascade distinct from β-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.
... Rab5-GTP localizes to the endosomal membrane, and Rab5-GDP is dispersed in the cytosol, and is associated with the Rab-GDP dissociation inhibitor (GDI) (2). Rab5 effectors, such as early-endosome antigen 1 (EEA1) and rabankyrin-5, bind not to Rab5-GDP, but to Rab5-GTP; thus a key determinant of Rab5 function is the duration of the GTP-bound state (3)(4)(5). In addition, approximately 20 cytosolic proteins specifically interact with active Rab5, suggesting a complex regulatory network (3). ...
The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides, and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus, and Homo sapiens, respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions.
... So far, Rabaptin5 had been characterized as a regulator of Rab5 activity by complex formation with Rabex5, the GDP/GTP exchange factor of Rab5. In addition it had been shown to bind to Rab4·GTP and Rab5·GTP, the hallmark Rab proteins of early endosomes (Horiuchi et al., 1997;Kälin et al., 2015;2016;Lippé et al., 2001;Mattera et al., 2006). Rabaptin5 thus was known to contribute to endosome identity and maturation (Huotari and Helenius, 2011). ...
Selective autophagy of damaged organelles is an important process for cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here we identified two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Indeed, autophagy of early endosomes damaged by chloroquine or monensin treatment was found to require Rabaptin5 and particularly a specific short sequence motif binding to the WD domain of ATG16L1. Rabaptin5 and this interaction with ATG16L1 is further required for much of autophagic elimination of Salmonella enterica in phagosomes with early endosomal characteristics early after infection. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes as a selective receptor to recruit autophagy to damaged early endosomes and phagosomes.
While the role of endocytosis in focal adhesion turnover-coupled cell migration has been established in addition to its conventional role in cellular functions, the molecular regulators and precise molecular mechanisms that underlie this process remain largely unknown. In this study, we report that proto-oncoprotein HPIP (hematopoietic PBX interacting protein) localizes to focal adhesions as well as endosomal compartments along with RUFY3 and Rab5, an early endosomal protein. HPIP contains two coiled-coil domains (CC1 and CC2) that are necessary for its association with Rab5 and RUFY3 as CC domain double mutant, i.e., mtHPIPΔCC1-2 failed to support it. Furthermore, we show that HPIP and RUFY3 activate Rab5 by serving as non-canonical guanine exchange factors (GEFs) of Rab5. In support of this, either deletion of coiled-coil domains or silencing of HPIP or RUFY3 impairs Rab5 activation and Rab5-dependent cell migration. Mechanistic studies further revealed that loss of HPIP or RUFY3 expression severely impairs Rab5-mediated focal adhesion disassembly, FAK activation, fibronectin (FN) associated-β1 integrin trafficking, and thus cell migration. Together, this study underscores the importance of HPIP and RUFY3 as non-canonical guanine exchange factors (GEFs) of Rab5, and in integrin trafficking and focal adhesion turnover, which implicates in cell migration.
Membranes are essential for life. They act as semi-permeable boundaries that define cells and organelles. In addition, their surfaces actively participate in biochemical reaction networks, where they confine proteins, align reaction partners, and directly control enzymatic activities. Membrane-localized reactions shape cellular membranes, define the identity of organelles, compartmentalize biochemical processes, and can even be the source of signaling gradients that originate at the plasma membrane and reach into the cytoplasm and nucleus. The membrane surface is, therefore, an essential platform upon which myriad cellular processes are scaffolded. In this review, we summarize our current understanding of the biophysics and biochemistry of membrane-localized reactions with particular focus on insights derived from reconstituted and cellular systems. We discuss how the interplay of cellular factors results in their self-organization, condensation, assembly, and activity, and the emergent properties derived from them.
Endosomal cargo recycling lies at the heart of subcellular trafficking processes under the management of several Ras-related GTP-binding proteins (Rabs) which are coordinated by their upstream regulators and require their downstream effectors to display their functions. In this regard, several Rabs have been well-reviewed except Rab22a. Rab22a is a crucial regulator of vesicle trafficking, early endosome and recycling endosome formation. Notably, recent studies demonstrated the immunological roles of Rab22a, which are closely associated with cancers, infection and autoimmune disorders. This review provides an overview of the regulators and effectors of Rab22a. Also, we highlight the current knowledge of the role of Rab22a in endosomal cargo recycling, including the biogenesis of recycling tubules with the help of a complex with Rab22a at its core, and how different internalized cargo chooses different recycling routes thanks to the cooperation of Rab22a, its effectors and its regulators. Of note, contradictions and speculation related to endosomal cargo recycling that Rab22a brings impacts on are also discussed. Finally, this review endeavors to briefly introduce the various events impacted by Rab22a, particularly focusing on the commandeered Rab22a-associated endosomal maturation and endosomal cargo recycling, in addition to the extensively investigated oncogenic role of Rab22a.
The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A combination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. Our study provides mechanistic insights into long-distance mRNA transport and demonstrates that the particular architecture of FERRY is closely linked to a previously undescribed mode of RNA binding, involving coiled-coil domains.
All G proteins contain a canonical GTPase fold for binding and hydrolyzing GTP, and consequently alternate between GTP- and GDP-bound conformations and can regulate diverse cellular functions. Small 20–30 kDa Ras-related G proteins include five families, i.e., Ras, Arf, Rab, Rho and Ran, and contain only the GTPase domain while large G proteins contain additional regulatory domains. The Rab family represents the largest branch of small G proteins involved in regulation of intracellular membrane trafficking in all eukaryotes from the last eukaryotic common ancestor (LECA) to human. In addition, other small and large G proteins discussed in this overview are essential regulators of signal transduction, cytoskeleton organization, and protein synthesis and translocation in the cell.
Phagosome maturation is an important innate defense mechanism of macrophages against pathogen infections. Phagosome‐lysosome (P‐L) fusion is a highly regulated process. Different RabGTPases are involved in P‐L fusion. Rab7l1 is shown to regulate P‐L fusion process. In the present study, we demonstrate that Rabaptin5 is a Guanine nucleotide exchange factor (GEF) for Rab7l1. We reveal that Rabaptin5 interacts with Rab7l1‐GTP form and promotes its recruitment to phagosome. In the absence of Rabaptin5, localization of P‐L markers like EEA1, Rab7, LAMP1 and LAMP2 was found to be poorer. Thus, our data suggest that Rabaptin5 works upstream to Rab7l1 and triggers Rab7l1 activation for further recruitment of P‐L markers and downstream regulation of phagosomal maturation process.
The multiligand receptors megalin and cubilin and their endocytic adaptor protein Dab2 play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells, and have complex and poorly understood roles in the development of chronic kidney disease. Here we used RNA-Seq and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin (Lrp2), cubilin (Cubn), or Dab2 (Dab2) expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in the Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggest changes in pathways with immunomodulatory functions that might trigger the pathologic changes observed in KO mice and in Donnai-Barrow patients. Additionally, differences in transcription patterns between Lrp2 and Dab2 KO cells suggest the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon disruption of PT endocytic function. A reduction in transcripts encoding Sglt2, was confirmed in Lrp2 KO mouse kidney lysates by qPCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.
Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluids into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines the fate of macropinosomes after they have been internalized is largely unknown. Here we investigate the role of the phosphatidylinositol 3-kinase VPS34/PIK3C3 and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. Inhibition of VPS34 led to a decrease in macropinosome survival and fluid phase uptake as well as preventing recruitment of early endosomal factors, including the small GTPase RAB5 and its effectors, to the forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A, and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane.
Whereas RAB5 was critical for macropinosome formation, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a PtdIns3P-controlled switch that balances macropinosome fate between the default, endolysosomal maturation and an alternative, secretory route.
His Domain Protein Tyrosine Phosphatase (HD-PTP) collaborates with Endosomal Sorting Complexes Required for Transport (ESCRTs) to sort endosomal cargo into intralumenal vesicles, forming the multivesicular body. Completion of multivesicular body sorting is accompanied by maturation of the endosome into a late endosome, an event that requires inactivation of the early endosomal GTPase, Rab5. Here we show that HD-PTP links ESCRT function with endosomal maturation. HD-PTP depletion prevents multivesicular body sorting, whilst also blocking cargo from exiting Rab5-rich endosomes. HD-PTP depleted cells contain hyperphosphorylated Rabaptin-5, a cofactor for the Rab5 guanine nucleotide exchange factor, Rabex-5, though HD-PTP is unlikely to directly dephosphorylate Rabaptin-5. In addition, HD-PTP depleted cells exhibit Rabaptin-5 dependent hyperactivation of Rab5. HD-PTP binds directly to Rabaptin-5, between its Rabex-5 and Rab5 binding domains. This binding reaction involves the ESCRT-0/ESCRT-III binding site in HD-PTP and is competed by an ESCRT-III peptide. Jointly, these findings indicate that HD-PTP may alternately scaffold ESCRTs and modulate Rabex-5/Rabaptin-5 activity, thereby helping to coordinate the completion of MVB sorting with endosomal maturation.
La Dystrophie Musculaire de Duchenne (DMD) est une maladie génétique causée par l’absence de dystrophine et provoquant une dégénérescence musculaire sévère. Aucun traitement curatif n’existe aujourd’hui mais la thérapie génique par vecteur AAV est l’une des stratégies les plus prometteuses pour traiter la DMD. Malgré l’efficacité bien établie de l’AAV de sérotype 8 (AAV8) pour le transfert de gène dans le muscle, de fortes doses de vecteurs sont nécessaires pour obtenir une efficacité thérapeutique dans des modèles animaux de la DMD. Dans ce contexte, j’ai étudié les mécanismes qui peuvent limiter l’efficacité de transduction du vecteur AAV8 dans le muscle dystrophique. Pour cela, j’ai étudié le devenir du vecteur AAV dans le muscle DMD puis caractérisé le système endosomal, essentiel au transport et la maturation des vecteurs AAV, dans différents modèles de la DMD. Mes résultats ont montré que l’efficacité de transduction de l’AAV8 est plus faible dans les cellules musculaires DMD comparées aux contrôles. De plus, la dérégulation du système endosomal dans la DMD peut impacter le transfert de gène par vecteur AAV dans ces cellules. Par ailleurs, l'amélioration de l'efficacité des vecteurs AAV en thérapie génique nécessite aussi une meilleure connaissance des protéines cellulaires qui interagissent avec le génome viral et qui régulent son expression. Dans ce contexte, nous avons montré que les facteurs de transcription RFX1 et RFX3 sont capables d’interagir avec la région ITR du génome viral et de moduler l’expression des vecteurs AAV.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) gives insight into the structure of proteins. By monitoring the rate of exchange of the amide hydrogens on the protein backbone with deuterium atoms in the solvent, one can determine if a given region is highly structured or dynamic, map binding sites of interacting molecules or determine if a binding event is associated with allosteric structural alterations in a protein. Herein, we illustrate the use of this technique to monitor the nucleotide exchange process in Rab5, using the guanine nucleotide exchange factor (GEF)–effector complex, Rabex5:Rabaptin5. By simultaneously monitoring the HDX in Rab5, Rabex5 and Rabaptin5, we can directly visualize nucleotide exchange in Rab5, gain mechanistic insights into the exchange reaction and, by witnessing the transfer of Rab5 from Rabex5 to Rabaptin5, provide direct evidence for the positive feedback loop generated by a GEF–effector complex. HDX-MS can be used to monitor a variety of Rab protein–effector and –regulator interactions and be widely applied to other enzymatic processes as well.
Endocytosis allows cells to transport particles and molecules across the plasma membrane. In addition, it is involved in the termination of signalling through receptor downmodulation and degradation. This traditional outlook has been substantially modified in recent years by discoveries that endocytosis and subsequent trafficking routes have a profound impact on the positive regulation and propagation of signals, being key for the spatiotemporal regulation of signal transmission in cells. Accordingly, endocytosis and membrane trafficking regulate virtually every aspect of cell physiology and are frequently subverted in pathological conditions. Two key aspects of endocytic control over signalling are coming into focus: context-dependency and long-range effects. First, endocytic-regulated outputs are not stereotyped but heavily dependent on the cell-specific regulation of endocytic networks. Second, endocytic regulation has an impact not only on individual cells but also on the behaviour of cellular collectives. Herein, we will discuss recent advancements in these areas, highlighting how endocytic trafficking impacts complex cell properties, including cell polarity and collective cell migration, and the relevance of these mechanisms to disease, in particular cancer. The canonical function of endocytosis is molecule internalization. Beyond this role, endocytic trafficking has emerged as a process central to the spatiotemporal regulation of cell signalling. Endocytic trafficking thus controls many cellular processes and tissue-wide properties, including cell migration and polarity, and its deregulation has been implicated in pathologies, particularly cancer.
L’élargissement des endosomes précoces (EP) a été amplement décrit dans la trisomie 21 (T21) et la maladie d’Alzheimer (MA). La présence d’EP élargis a été démontrée dans des neurones avant même le développement de la neuropathologie et des symptômes de la MA. L’élargissement des EP a également été identifié dans de nombreux modèles de MA et de T21. Il a été proposé que la surexpression d’APP induise l’élargissement des EP, mais certaines observations suggèrent une origine multifactorielle à l’élargissement des EP. Malgré la petite taille des EP, leur morphologie n’a jamais été analysée à haute résolution. Dans cette étude, nous caractérisons les EP et la voie endosomale dans la T21 afin de mieux en comprendre les phénotypes et les mécanismes moléculaires associés. Grâce à différentes méthodes de microscopie électronique et super-résolutive, nous observons dans de nombreux modèles de T21 une augmentation du nombre d’EP de taille normale ayant tendance à s’agréger plutôt que des EP élargis. Nous montrons que le recyclage du récepteur à la transferrine est augmenté alors que la dégradation du récepteur à l’EGF est ralentie. Nous montrons ensuite que les niveaux de Rab5 et de PI(3)P sont augmentés et le niveau d’EEA1 est diminué. Enfin, l’inhibition de la kinase PIKfyve permet de rétablir le niveau d’EEA1 et la production d’Aβ dans des fibroblastes T21. Notre étude redéfinit les phénotypes de la voie endo-lysosomale dans la T21 aux niveaux morphologiques, dynamiques et moléculaires. Ces résultats apportent de nouvelles hypothèses mécanistiques pour expliquer les anomalies endosomales dans la T21 et la MA.
Alzheimer's disease (AD) is the most common form of dementia and the most prevalent neurodegenerative disease. Genome Wide Association Studies have linked PICALM to AD risk. PICALM has been implicated in Aβ42 production and turn-over, but whether it plays a direct role in modulating Aβ42 toxicity remains unclear. We found that increased expression of the Drosophila PICALM orthologue lap could rescue Aβ42 toxicity in an adult-onset model of AD, without affecting Aβ42 level. Imbalances in the glutamatergic system, leading to excessive, toxic stimulation have been associated with AD. We found that Aβ42 caused accumulation of pre-synaptic vesicular glutamate transporter (VGlut) and increased spontaneous glutamate release. Increased lap expression reversed these phenotypes back to control levels, suggesting that lap may modulate glutamatergic transmission. We also found that lap modulated the localisation of Amph, the homologue of another AD risk factor BIN1, and that Amph itself modulated post-synaptic glutamate receptor (GluRII) localisation. We propose a model where PICALM modulates glutamatergic transmission, together with BIN1, to ameliorate synaptic dysfunction and disease progression.
We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.
Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.
A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein.
Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes
and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein,
which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras).
Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding
by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These
results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates
only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity,
and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence
characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein,
Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A
GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.
Small GTPases of the Rab family are key regulators of intracellular transport. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecting these compartments. Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibitor (RabGDI). Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form. In this paper we have investigated whether Rab5, which regulates the clathrin-coated vesicle-mediated pathway of endocytosis, can directly associate with the membrane of clathrin-coated vesicles (CCV) purified from bovine brain in vitro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV. Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociation of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP. The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone. However, maximal GDP dissociation stimulation required the presence of RabGDI, suggesting that the factor(s) responsible for the membrane association and GDP/GTP exchange of Rab5 recognize the protein complexed to RabGDI. These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.
To identify potential Sec4 effectors, we isolated high copy suppressors of a Sec4 effector domain mutant. The most potent of these was found to be SEC9, a gene required for post-Golgi transport. The sole essential domain of Sec9 has significant sequence similarity to the neuronal protein SNAP-25, a component of the SNARE complex, that is implicated in vesicle targeting and fusion. Analogous to SNAP-25, Sec9 is bound to the yeast plasma membrane and is absent from post-Golgi vesicles. Furthermore, Sec9 is physically associated with two proteins that are homologous to components of the neuronal SNARE complex. Our results identify Sec9 as the yeast cognate of SNAP-25 and suggest that SNARE complexes acting at specific stages of vesicular transport serve as the ultimate targets of regulation by members of the Sec4/Ypt1/Rab family of GTPases.
Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4-GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co-precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.
The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.
The Rab GTPases are key regulators of vesicular transport. A fraction of Rab proteins is present in the cytosol, bound with GDP, complexed to a protein termed GDI. Rab9 is localized primarily to late endosomes, where it aids the transport of mannose 6-phosphate receptors to the trans-Golgi network. It has been proposed that Rab proteins are delivered to specific membranes by GDI, and that this process is accompanied by the exchange of bound GDP for GTP. In addition, Rab localization requires carboxy-terminal prenylation and specific structural determinants. Here we describe the reconstitution of the selective targeting of prenylated Rab9 protein onto late endosome membranes and show that this process is accompanied by endosome-triggered nucleotide exchange.
Rab5 is a Ras-like small GTPase that regulates early events of endocytosis. Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants. Second-site mutations were introduced into Rab5:S34N and Rab5:N133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N133I. The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras. This mutation greatly decreased the isoprenylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector domain Phe57 residue and C-terminal isoprenylation of Rab5.
In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.
We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport. It is mainly cytosolic, but a fraction colocalizes with Rab5 to early endosomes. Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged endosomes. Overexpression of rabaptin-5 alone is sufficient to promote expansion of early endosomes. Rab5 recruits rabaptin-5 to purified early endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily. Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion. Rabaptin-5 is thus a Rab effector required for membrane docking and fusion.
Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.
Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.
S. cerevisiae inherits its vacuole by projecting vacuole-derived membrane vesicles and tubules into the bud, where they fuse to establish the daughter vacuole. This homotypic fusion event can be assayed in vitro. It requires Sec17p and Sec18p, the homologs of the mammalian alpha-SNAP and NSF, which cooperate in multiple steps of membrane trafficking. We now report that Sec17p, Sec18p, and ATP are only needed for an early stage of the reaction that results in Sec17p release. Sec17p and Sec18p actions precede, and are needed for, the step employing the Ras-like GTPase Ypt7p. Sec18p-driven release of Sec17p can even precede vacuole docking, as it can occur prior to mixing of vacuoles and is insensitive to vacuole concentration. Sec17p and Sec18p thus may function in a predocking stage of the reaction, rather than in bilayer fusion per se.
In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.
Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.
We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta-repeat domains characteristic of the beta-subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid-rich regions, a putative leucine zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy-terminal RCC1-like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1-like motif located at the amino-terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R-Ras2/TC21. These observations suggest that p619 is a Brefeldin A-sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.
Vacuole inheritance in yeast involves the formation of tubular and vesicular "segregation structures" which migrate into the bud and fuse there to establish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and microscopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacuoles must be primed for docking by the action of Sec17p (alpha-SNAP) and Sec18p (NSF). Priming is necessary for both fusion partners. It produces a labile state which requires rapid docking in order to lead productively to fusion. In addition to Sec17p/Sec18p, docking requires the activity of the Ras-like GTPase Ypt7p. Unlike Sec17p/Sec18p, which must act before docking, Ypt7p is directly involved in the docking process itself.
The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They
cycle between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of
a GDP/GTP exchange protein (GEP). No GEP specific for each Rab family member or Rab subfamily has been isolated. Here we purified
a GEP from rat brain with lipid-modified Rab3A as a substrate. The purified protein was specifically active on Rab3A, Rab3C,
and Rab3D of the Rab3 subfamily. Of these subfamily members, Rab3A and Rab3C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release. This GEP (Rab3 GEP) was active on the lipid-modified form,
but not on the lipid-unmodified form. Rab3 GEP showed a minimum molecular mass of about 200 kDa on SDS-polyacrylamide gel
electrophoresis. We cloned its cDNA from a rat brain cDNA library and determined its primary structure. The isolated cDNA
encoded a protein with a Mr of 177,982 and 1,602 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GEP activity
toward Rab3A, Rab3C, and Rab3D. Northern blot and Western blot analyses indicated that Rab3 GEP was expressed in all the rat
tissues examined with the highest expression in brain.
Prenylated Rab GTPases occur in the cytosol in their GDP-bound conformations bound to a cytosolic protein termed GDP-dissociation inhibitor (GDI). Rab-GDI complexes represent a pool of active, recycling Rab proteins that can deliver Rabs to specific and distinct membrane-bound compartments. Rab delivery to cellular membranes involves release of GDI, and the membrane-associated Rab protein then exchanges its bound GDP for GTP. We report here the identification of a novel, membrane-associated protein factor that can release prenylated Rab proteins from GDI. This GDI-displacement factor (GDF) is not a guanine nucleotide exchange factor because it did not influence the intrinsic rates of nucleotide exchange by Rabs 5, 7 or 9. Rather, GDF caused the release of each of these endosomal Rabs from GDI, permitting them to exchange nucleotide at their intrinsic rates. GDF displayed the greatest catalytic rate enhancement on Rab9-GDI complexes. However, catalytic rate enhancement paralleled the potency of GDI in blocking nucleotide exchange: GDI was shown to be most potent in blocking nucleotide exchange by Rab9. The failure of GDF to act on Rab1-GDI complexes suggests that it may be specific for endosomal Rab proteins. This novel, membrane-associated activity may be part of the machinery used to localize Rabs to their correct intracellular compartments.
The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2,
itself an essential gene that acts at the same stage of the
secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation
of vesicles. Sec2p and Sec4p interact directly, with the
nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions
as an exchange protein, catalyzing the dissociation of
GDP from Sec4 and promoting the binding of GTP. We
propose that Sec2p functions to couple the activation of
Sec4p to the polarized delivery of vesicles to the site of
exocytosis.
In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.
Transport between Golgi dsternae in a cell-free system is blocked hy cither nonhydrolyzable analogue ol'GTP (GTPyS) or by the sulfhydryl reagenl N-cIhylmaleimide (NEM). With GTPyS, vesicles accumulate encased in a coat which is fro/en in place because GTP cannot he hydrnlyzed. With NEM, uncoated vesicles accumulate ducked to their target cisterna. but tail to fuse; NEM inactivates a protein required tor fusion, termed NSF, for NEM Sensitive Fusion protein. The coal is composed uf many copies of a protein termed coatomcr (short for coat protomer), an assembly of seven distinct subunils. termed COP proteins. The coated vesicles also contain many copies of ;i GTPase, ARF. Cytosohc ARF binds GTP, binds to the Golgi, and then recruits cytosolic coatomer lo join the assembling coat. Later, after budding, ARF hydrolyzes its bound GTP, releasing coatomer; this explains why non-hydrolyzable GTP blocks transport and accumulates coated transport vesicles. Similar or identical COPI-coated vesicles mediate antcrograde and retrograde transport both in isolated Golgi stacks ;md in whole cells.
The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.
Fusion between endocytic vesicles containing 125I-transferrin and ones containing anti-transferrin antibody was monitored by the formation of an immune complex. Fusion required ATP and only occurred between specific populations of vesicles. Cytosol was also required. Trypsinisation of the preparations or pre-treatment with N-ethylmaleimide abolished the fusion reaction.
The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.
The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.
Rabphilin3a had been identified in brain as a Rab3a-binding protein and may serve as an effector for Rab3a function. We have cloned a splice variant of brain-Rabphilin3a from a bovine adrenal chromaffin cell cDNA library and investigated the function of the protein in regulated exocytosis in bovine chromaffin cells. The predicted amino acid sequence of chromaffin cell (c-) Rabphilin3a was identical with that of brain (b-) Rabphilin3a except for a 6-amino-acid insert VFSLSA in the amino-terminal half of the protein. An antibody directed against a carboxyl-terminal peptide recognized an 85-kDa protein in COS7 cells transfected with the cDNA in a mammalian expression vector. A band of similar mobility was enriched in a fraction of highly purified chromaffin granule membranes, consistent with the Rabphilin3a being associated with chromaffin granule membranes. Overexpression of either chromaffin cell or brain Rabphilin3a by transfection with the corresponding cDNAs in mammalian expression vectors enhanced DMPP-induced secretion of co-expressed human growth hormone (GH) approximately 30%. Chromaffin cells transfected with a plasmid with the entire coding sequence of c-Rabphilin3a inserted in the antisense orientation inhibited secretion of co-expressed GH by approximately 30%. Rabphilin3a mutants lacking one or both of the carboxyl-terminal C2 domains strongly inhibited DMPP-stimulated exocytosis. The single C2 domain deletion also strongly inhibited Ca(2+)-dependent secretion from digitonin-permeabilized cells. These data indicate that Rabphilin3a is a positive regulator of exocytosis. Because the C2 deletion mutants contain the amino-terminal Rab3a-GTP binding domain, they may inhibit secretion by competing with endogenous Rabphilin3a for interaction with Rab3a-GTP without being able to mimic the functional effects of full-length Rabphilin3a.
The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces
cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating
mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein
encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named
this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP.
Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a
human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of
Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides
primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells
as an effector or regulator of H-Ras.
The SNARE hypothesis provides a mechanism for the specific docking and fusion of transport vesicles with their target membranes. A simple extension of the hypothesis can explain many cellular processes, including the stacking of Golgi cisternae, retrograde transport and homotypic fusion; it can also explain the morphology of intracellular membranes and their dynamics during mitosis.
Rab proteins are generally required for transport vesicle docking. We have exploited yeast secretion mutants to demonstrate that a rab protein is required for v-SNAREs and t-SNAREs to assemble. The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly. In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking. This mechanism likely involves the Sec1 homolog Sly1, which we identified in isolated docking complexes. We also report the identification of a novel v-SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has a CAAX box and is predicted to be lipid anchored. The surprising finding that docking complexes can contain many distinct species of SNAREs (Sed5p, Bos1p, Sec22p, Ykt6p, and likely Bet1p, p28, and p14) suggests that multimeric interactions are features of the fusion machinery, and may also improve the fidelity of vesicle targeting.
Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.
Rab GTPases are thought to be likely to catalyze the accurate association of pairs of targeting molecules located on the surfaces of transport vesicles with their corresponding membrane acceptors. Advances during the past year have solidified our understanding of the mechanisms by which Rab proteins are recruited onto nascent transport vesicles and retrieved from their fusion targets. Functional analyses of Rab proteins in living cells have led to the surprising observation that vesicles do not seem to form if the appropriate Rab protein, in its GTP-bound conformation, is not present.
Synaptobrevin-like membrane proteins that reside on transport vesicles, called the vesicle SNARE (v-SNARE), play a key role in ensuring that a vesicle targets and fuses with its correct acceptor compartment. Here we show that Bos1p, the v-SNARE of yeast endoplasmic reticulum-to-Golgi transport vesicles, pairs with another integral membrane protein of similar topology (Sec22p) on vesicles. This pairing, which appears to require functional Ypt1p (Rab in mammalian cells), may aid the activity of Bos1p on this compartment. These findings suggest that Rabs regulate the specificity of membrane fusion by selectively activating the v-SNARE on carrier vesicles. Because the v-SNARE resides on more than one membrane, such a regulated activation step may be necessary to prevent the premature fusion of donor and acceptor compartments.
Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.
The Rab GTPases function as specific regulators of membrane transport. The GTP/GDP cycle is believed to control shuttling of Rab proteins between the cytosol and organelle membranes. In vitro, Rab proteins are removed from membranes by a protein that inhibits GDP dissociation (rabGDI), which leads to formation of a cytosolic complex of Rab with the inhibitor protein. Here we use a purified Rab5-rabGDI complex in a permeabilized cell system to investigate how the cytosolic complexed form of Rab reassociates with the membrane. We find that exogenous Rab5 is correctly targeted and induces the formation of enlarged early endosomes, demonstrating that it is functionally active. Binding of Rab5 to the acceptor membrane is accompanied by release of the rabGDI protein into the cytosol. A transient GDP-Rab5 intermediate was detected which was subsequently converted into the GTP-bound form. Our results indicate that there is a multistep mechanism for the insertion of Rab5 into the membrane which is mediated by a guanine-nucleotide-exchange factor.
The SNARE hypothesis holds that a transport vesicle chooses its target for fusion when a soluble NSF attachment protein (SNAP) receptor on the vesicle (v-SNARE) pairs with its cognate t-SNARE at the target membrane. Three synaptosomal membrane proteins have previously been identified: syntaxin, SNAP-25 (t-SNAREs), and vesicle-associated membrane protein (VAMP) (v-SNARE); all assemble with SNAPs and NSF into 20S fusion particles. We now report that in the absence of SNAP and NSF, these three SNAREs form a stable complex that can also bind synaptotagmin. Synaptotagmin is displaced by alpha-SNAP, suggesting that these two proteins share binding sites on the SNARE complex and implying that synaptotagmin operates as a "clamp" to prevent fusion from proceeding in the absence of a signal. The alpha-SNAP-SNARE complex can bind NSF, and NSF-dependent hydrolysis of ATP dissociates the complex, separating syntaxin, SNAP-25, and VAMP. ATP hydrolysis by NSF may provide motion to initiate bilayer fusion.
Specificity and directionality are two features shared by the numerous steps of membrane transport that connect cellular organelles. By shuttling between specific membrane compartments and the cytoplasm, small GTPases of the Rab family appear to regulate membrane traffic in a cyclical manner. The restriction of certain Rab proteins to differentiated cell types supports a role for these GTPases in defining the specificity of membrane trafficking.
Small GTP-binding proteins of the ras superfamily are important for exocytosis from eukaryotic cells. GTP-binding proteins can exist in two different conformations depending on whether they are bound to GDP or GTP, and are thought to function as molecular switches that regulate a variety of cellular processes. The GTP-GDP cycle is controlled by accessory proteins that promote the exchange of bound GDP or the hydrolysis of GTP. The protein Sec4, a member of the Sec4/Ypt1/Rab branch of the Ras superfamily, is involved in a late stage of the secretory pathway in yeast. Here we report the isolation of a mammalian complementary DNA, mss4, encoding a GDP-releasing protein that enhances Sec4 function. The Mss4 protein also stimulates GDP release from Ypt1 and from the mammalian protein Rab3a, but not from Ras2. Mss4 shows sequence similarity to Dss4, a yeast protein with similar biochemical properties.
The protein Sec4p plays an essential role at the final stage of the yeast secretory pathway and belongs to the ras superfamily of GTP-binding proteins, more specifically to a branch that includes Ypt1p in Saccharomyces cerevisiae and rab proteins in mammalian cells. GTP-binding proteins change conformation depending on whether GTP or GDP is bound and can thus act as a regulatory switch. The protein remains in its inactive, GDP-bound form until exchange of GTP for GDP allows it to stimulate a downstream effector. This interaction is curtailed by GTP hydrolysis. The rates of nucleotide exchange and GTP hydrolysis can be regulated by interaction with accessory proteins. Although GDP dissociation stimulators (GDS) have been identified that act on members of the ras and rho branches of the superfamily, less is known regarding GDSs that act on members of the Sec4/Ypt1/Rab subgroup. A preliminary characterization of a Rab3A GDP dissociation stimulating activity has been presented. We report here the use of suppressor analysis to clone a gene, dss4, encoding a 17K protein that aids Sec4p action in vivo by functioning as a GDP dissociation stimulator.
Rab5 is a 25-kDa GTP-binding protein localized to the plasma membrane, clathrin-coated vesicles, and early endosomes, and it functions as a regulatory factor of endocytosis. As for other Rab proteins, Rab5 is geranylgeranylated at its C terminus, and this modification is essential for its function. To obtain Rab5 in the isoprenylated form, the chapter makes use of a baculovirus expression system. This chapter describes a method to purify both posttranslationally modified and unmodified Rab5 from Spodoptera frugiperda (Sf9) insect cells overexpressing the protein. Purified posttranslationally modified and unmodified Rab5 protein efficiently binds GTP and GDP. However, as expected, Rab-GDI is active only on modified Rab5. When modified Rab5 complexed with Rab-GDI is introduced into permeabilized cells, Rab5 is localized to its correct site of function and induces the formation of enlarged early endosomes as previously observed in vivo, indicating that it is functionally active.
This chapter describes the permeabilized cell system and the methods used to study the role of Rab-GDI in the reversible membrane association of Rab proteins. The advantage of using permeabilized cells is that, while the cellular organization is maintained, the intracellular membranes are accessible to biochemical manipulations. Thus, this in vitro system closely mimics the physiological conditions in intact cells. Rab proteins are associated with specific compartments along the exocytic and endocytic pathways. Membrane association requires posttranslational modification of the C-terminal cysteines by the addition of geranylgeranyl moieties. In addition, Rab proteins are also present in the cytosol. Cytosolic Rab proteins are complexed to Rab-GDI (GDP dissociation inhibitor), a factor that inhibits the dissociation of GDP and removes Rab proteins from the membrane. These biochemical properties have led to the suggestion that Rab-GDI allows Rab proteins to shuttle between the membrane and the cytosol to serve multiple rounds of vesicular traffic.
Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
The nanoelectrospray ion source (nanoES) has recently been developed and described theoretically. It is different from conventional electrospray sources and from other miniaturized electrospray sources by (i) its 1-2 microns spraying orifice achieved by pulling the spraying capillary to a fine tip, (ii) its very low flow rate of approximately 20 nL/min and the small size of droplets it generates, and (iii) the absence of solvent pumps and inlet valves. The fabrication and operation of nanoES needles is described in detail. Solutions with up to 0.1 M salt contents could be sprayed without sheath flow or pneumatic assist. Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein. Extensive mass spectrometric and tandem mass spectrometric investigation of the components of an unseparated peptide mixture was demonstrated by verification of 93% of the sequence of carbonic anhydrase. A rapid and robust desalting/concentration step coupled to the nanoES procedure allows the direct analysis of impure samples such as peptide mixtures extracted after in-gel digestion.
The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport. Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein. To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5. Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.
Intracellular vesicle targeting involves the interaction of vesicle proteins, termed v-SNAREs, with target membrane proteins,
termed t-SNAREs. Assembly of v-SNARE–t-SNARE targeting complexes is modulated by members of the Sec1-Sly1 protein family,
and by small guanosine triphosphatases termed Rabs. The interactions of these proteins during assembly of the endoplasmic
reticulum–to-Golgi targeting complex inSaccharomyces cerevisiae were studied. The data suggest that the Rab protein Ypt1p transiently interacts with the t-SNARE Sed5p and results in displacement
of the negative regulator Sly1p, allowing subsequent formation of the v-SNARE–t-SNARE targeting complex.
Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.