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Working model for the coupling of vacuolar priming, docking, and trans-SNARE pairing. v, t, and s symbolize the v-SNAREs (Nyv1p, Vti1p, and Ykt6p), the t-SNARE Vam3p, and the SNAP-25 homologue Vam7p, respectively. Y7, Ypt7p; 2/6, Vam2/6 complex. See text for details.
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The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the...
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Background:
The yeast Pichia pastoris is a widely used host for the secretion of heterologous proteins. Despite being an efficient producer, we observed previously that certain recombinant proteins were mistargeted to the vacuole on their route to secretion. Simultaneous disruption of one vacuolar sorting pathway together with vacuolar proteases p...
Citations
... HOPS is a complex of six subunits, four 33 of them shared (VPS11, VPS33, VPS16 and VPS18) with CORVET, another complex involved in 34 endosome life cycle. The remaining two, VPS39 and VPS41 are required for the tethering function, 35 with VPS41 the effector subunit of HOPS promoting vacuole fusion (Price et al., 2000). VPS41 is 36 localized in late endosomes in Arabidopsis (Arabidopsis thaliana) (Brillada et al., 2018). ...
Resistance to Cucumber mosaic virus (CMV) in melon (Cucumis melo L.) has been described in several exotic accessions and is controlled by a recessive resistance gene, cmv1, that encodes a Vacuolar protein sorting 41 (CmVPS41). cmv1 prevents systemic infection by restricting the virus to the bundle sheath cells, preventing viral phloem entry. CmVPS41 from different resistant accessions carries two causal mutations, either a G85E change, found in Pat-81 and Freeman’s Cucumber, or L348R, found in PI161375, cultivar Songwhan Charmi (SC). Here, we analyzed the subcellular localization of CmVPS41 in Nicotiana benthamiana and found differential structures in resistant and susceptible accessions. Susceptible accessions showed nuclear and membrane spots and many transvacuolar strands, whereas the resistant accessions showed many intravacuolar invaginations. These specific structures colocalized with late endosomes. Artificial CmVPS41 carrying individual mutations causing resistance in the genetic background of CmVPS41 from the susceptible variety Piel de Sapo (PS) revealed that the structure most correlated with resistance was the absence of transvacuolar strands. Co-expression of CmVPS41 with viral MPs, the determinant of virulence, did not change these localizations; however, infiltration of CmVPS41 from either SC or PS accessions in CMV-infected N. benthamiana leaves showed a localization pattern closer to each other, with up to 30% cells showing some membrane spots in the CmVPS41SC and fewer transvacuolar strands (reduced from a mean of 4 to 1-2) with CmVPS41PS. Our results suggest that the distribution of CmVPS41PS in late endosomes includes transvacuolar strands that facilitate CMV infection and that CmVPS41 re-localizes during viral infection.
... Indeed, the continuous activity of NSF appears to be required to maintain SNAREs in an active form since they not only form complexes during fusion (trans-assembly) but also have a tendency to form cis-complexes in the membrane [38]. For instance, studies on yeast homotypic vacuolar fusion in vitro have revealed that Sec18 (the yeast homologue of mammalian NSF) and Sec17 (the yeast homologue of mammalian α-SNAP) are necessary for subsequent tethering and fusion [39][40][41]. NSF and SNAPs not only operate on fully assembled cis-complexes but also dissociate partial complexes. Moreover, recent evidence suggests that binding of α-SNAP, and perhaps also NSF, may also occur to SNARE trans-complexes and facilitate fusion without requiring ATP hydrolysis [42]. ...
Membrane traffic in eukaryotic cells is mediated by transport vesicles that bud from a precursor compartment and are transported to their destination compartment where they dock and fuse. To reach their intracellular destination, transport vesicles contain targeting signals such as Rab GTPases and polyphosphoinositides that are recognized by tethering factors in the cytoplasm and that connect the vesicles with their respective destination compartment. The final step, membrane fusion, is mediated by SNARE proteins. SNAREs are connected to targeting signals and tethering factors by multiple interactions. However, it is still debated whether SNAREs only function downstream of targeting and tethering or whether they also participate in regulating targeting specificity. Here, we review the evidence and discuss recent data supporting a role of SNARE proteins as targeting signals in vesicle traffic.
... Extensive biochemistry studies have also led to a comprehensive characterization of the many conserved protein families universally involved in these trafficking events [reviewed by Rizo and Rosenmund (2008), Sudhof and Rizo (2011), Rizo and Sudhof (2012), and Rizo and Xu (2015)]. Each trafficking event requires specific biochemical interactions between proteins of SVs, AZM macromolecules, and the PM to proceed (Takamori et al., 2006;Südhof and Rothman, 2009;Südhof, 2012Südhof, , 2013Snead and Eliezer, 2019), although the mechanistic details of each event are under considerable debate (Hanson et al., 1997;Jahn and Sudhof, 1999;Klenchin and Martin, 2000;Price et al., 2000;Jahn et al., 2003;Szule and Coorssen, 2003;Han et al., 2004;Südhof, 2004;Jackson and Chapman, 2008;Neher and Sakaba, 2008;Chua et al., 2010;Gundersen and Umbach, 2013;Szule et al., 2015). A hypothesized proteomic atlas will be provided here to describe how these conserved proteins are thought to be assembled and function in their AZM macromolecular complexes in situ to regulate SV docking, priming, Ca 2+ -triggering, and membrane fusion that ultimately control the regulation of triggered neurotransmitter secretion. ...
... Syntaxin, SNAP25 with Synaptobrevin together referred to as SNARE proteins (Soluble NSF-Attachment Protein Receptor) assemble to form the SNARE complex, and several models have implicated the complex as essential for membrane fusion (Sollner et al., 1993b;Weber et al., 1998;Melia et al., 2002;Han et al., 2004;Südhof and Rothman, 2009;Jackson, 2010;Karatekin et al., 2010). Additionally, other models suggest the roles of the SNARE complex to be upstream to membrane fusion, such as during docking and priming Tahara et al., 1998;Price et al., 2000;Harlow et al., 2001Harlow et al., , 2013Coorssen, 2003, 2004;Szule et al., , 2012Gundersen and Umbach, 2013;Imig et al., 2014;Meriney et al., 2014;Jung et al., 2016). ...
This report integrates knowledge of in situ macromolecular structures and synaptic protein biochemistry to propose a unified hypothesis for the regulation of certain vesicle trafficking events (i.e., docking, priming, Ca ²⁺ -triggering, and membrane fusion) that lead to neurotransmitter secretion from specialized “active zones” of presynaptic axon terminals. Advancements in electron tomography, to image tissue sections in 3D at nanometer scale resolution, have led to structural characterizations of a network of different classes of macromolecules at the active zone, called “Active Zone Material’. At frog neuromuscular junctions, the classes of Active Zone Material macromolecules “top-masts”, “booms”, “spars”, “ribs” and “pins” direct synaptic vesicle docking while “pins”, “ribs” and “pegs” regulate priming to influence Ca ²⁺ -triggering and membrane fusion. Other classes, “beams”, “steps”, “masts”, and “synaptic vesicle luminal filaments’ likely help organize and maintain the structural integrity of active zones. Extensive studies on the biochemistry that regulates secretion have led to comprehensive characterizations of the many conserved proteins universally involved in these trafficking events. Here, a hypothesis including a partial proteomic atlas of Active Zone Material is presented which considers the common roles, binding partners, physical features/structure, and relative positioning in the axon terminal of both the proteins and classes of macromolecules involved in the vesicle trafficking events. The hypothesis designates voltage-gated Ca ²⁺ channels and Ca ²⁺ -gated K ⁺ channels to ribs and pegs that are connected to macromolecules that span the presynaptic membrane at the active zone. SNARE proteins (Syntaxin, SNAP25, and Synaptobrevin), SNARE-interacting proteins Synaptotagmin, Munc13, Munc18, Complexin, and NSF are designated to ribs and/or pins. Rab3A and Rabphillin-3A are designated to top-masts and/or booms and/or spars. RIM, Bassoon, and Piccolo are designated to beams, steps, masts, ribs, spars, booms, and top-masts. Spectrin is designated to beams. Lastly, the luminal portions of SV2 are thought to form the bulk of the observed synaptic vesicle luminal filaments. The goal here is to help direct future studies that aim to bridge Active Zone Material structure, biochemistry, and function to ultimately determine how it regulates the trafficking events in vivo that lead to neurotransmitter secretion.
... Ras-associated binding GTPases (Rab) function in multiple steps of membrane trafficking, including protein sorting and vesicle transport [14]. For the fusion process of yeast vacuoles, the homotypic fusion and vacuole protein sorting (HOPS) complex is initially associated with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and then complexed with Rab GTPase Ypt7 for tethering and vacuole membrane fusion [15,16]. ...
The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress ( sod1 Δ) and vacuolar and protein sorting ( vps1 Δ and ypt7 Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12 Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O . thermomethanolica and used it to activate SOD1 , VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O . thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.
... The fusion of late endosomes and lysosomes is dependent on the HOPS complex, which consists of six subunits, VPS11, 16, 18, 33, 39, and 41 (Balderhaar and Ungermann, 2013;Bröcker et al., 2012;Graham et al., 2013). In yeast, Vps39 and 41 directly interact with Ypt7/Rab7, enabling the HOPS complex to tether late endosomes and lysosomes for fusion (Price et al., 2000). Because the lysosomal function of WDR91 requires its interaction with Rab7, we examined whether WDR91 interacts with VPS39 and/ or VPS41. ...
The effectors of the Rab7 small GTPase play multiple roles in Rab7-dependent endosome-lysosome and autophagy-lysosome pathways. However, it is largely unknown how distinct Rab7 effectors coordinate to maintain the homeostasis of late endosomes and lysosomes to ensure appropriate endolysosomal and autolysosomal degradation. Here we report that WDR91, a Rab7 effector required for early-to-late endosome conversion, is essential for lysosome function and homeostasis. Mice lacking Wdr91 specifically in the central nervous system exhibited behavioral defects and marked neuronal loss in the cerebral and cerebellar cortices. At the cellular level, WDR91 deficiency causes PtdIns3P-independent enlargement and dysfunction of lysosomes, leading to accumulation of autophagic cargoes in mouse neurons. WDR91 competes with the VPS41 subunit of the HOPS complex, another Rab7 effector, for binding to Rab7, thereby facilitating Rab7-dependent lysosome fusion in a controlled manner. WDR91 thus maintains an appropriate level of lysosome fusion to guard the normal function and survival of neurons.
... It was found that loss of CmVps39 also made the vacuoles smaller in Coniothyrium minitans. Vacuoles can be formed by aggregation of small vesicles to form large vacuoles, or by split of large vacuoles in C. albicans [15,17,37]. Fusion and division of vacuoles were indispensable to maintain normal cell viability [38]. ...
Candida albicans is one of the most common opportunistic fungal pathogens in human beings. When infecting host cells, C. albicans is often exposed to oxidative stress from the host immune defense system. Maintenance of mitochondrial and vacuolar functions is crucial for its resistance to oxidative stress. However, the role of vacuole and mitochondria patchs (vCLAMPs) in cellular oxidative stress resistance and in the maintenance of organelle functions remains to be elucidated. Herein, the function of the vCLAMP protein Vam6 in response to oxidative stress was explored. The results showed that the vam6∆/∆ mutant exhibited obvious mitochondrial swelling, mtDNA damage, reduced activity of antioxidant enzymes, and abnormal vacuolar morphology under H2O2 treatment, indicating its important role in maintaining the structures and functions of both mitochondria and vacuoles under oxidative stress. Further studies showed that deletion of VAM6 attenuated hyphal development under oxidative stress. Moreover, loss of Vam6 obviously affected host tissue invasion and virulence of C. albicans. Taken together, this paper reveals the critical role of vCLAMPs in response to oxidative stress in C. albicans.
... CORVET contains Vps3p and Vps8p, which bind to the Rab5 homolog, Vps21p, found on early endosomal membranes [26,27]. HOPS complex contains Vps39p and Vps41p, which bind to the Rab7 homolog, Ypt7p, found on late endosomal and vacuolar membrane compartments [28][29][30]. As both HOPS and CORVET complexes are disrupted in class C core mutant strains, we individually deleted each of the Rab-specific subunits of these complexes to determine the effects of CORVET-or HOPS-specific disruptions on LegC7 activity. ...
Legionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires’ disease. Legionella actively alters host organelle trafficking through the activities of ‘effector’ proteins secreted via a TypeIVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is derived from membrane derived from host ER, secretory vesicles, and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology, and induces aberrant ER:endosome fusion, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins, and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology, and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by inducing membrane fusion, potentially through interaction with host membrane tethering complexes and/or cargo adapters.
... The process of membrane tethering is vital for determining the spatiotemporal specificity of intracellular membrane trafficking, before the irreversible final steps of membrane docking and fusion mediated by SNARE-family proteins (Jahn and Scheller, 2006), which are another critical layers to confer the fidelity of membrane trafficking (McNew et al., 2000;Parlati et al., 2002;Izawa et al., 2012;Furukawa and Mima, 2014). A large body of prior studies on membrane tethering or vesicle tethering (or capture) have identified a number of the protein components essential for membrane tethering (Yu and Hughson, 2010;Kuhlee et al., 2015;Cheung and Pfeffer, 2016;Spang, 2016;Witkos and Lowe, 2016;Gillingham and Munro, 2019), which include the Uso1/p115 coiled-coil protein (Sapperstein et al., 1995(Sapperstein et al., , 1996Barlowe, 1997;Cao et al., 1998), golgin family coiled-coil proteins (Drin et al., 2008;Wong and Munro, 2014;Cheung et al., 2015), the EEA1 coiled-coil protein (Murray et al., 2016), and a diversified set of multisubunit tethering complexes, such as the HOPS complex (Price et al., 2000;Stroupe et al., 2009;Hickey and Wickner, 2010;Stroupe, 2015, 2016), the exocyst complex (TerBush et al., 1996;Guo et al., 1999;Rossi et al., 2020), the COG complex (Ungar et al., 2002;Zolov and Lupashin, 2005;Shestakova et al., 2006), the Dsl1 complex (Reilly et al., 2001;Ren et al., 2009;Zink et al., 2009), the GARP complex (Conibear and Stevens, 2000;Pérez-Victoria et al., 2008;Pérez-Victoria and Bonifacino, 2009), and the TRAPP complex (Sacher et al., 2001;Cai et al., 2005Cai et al., , 2007. It is noteworthy that, in addition to these miscellaneous, sequentiallyand structurally-diverse classic tethering factors, our recent reconstitution studies have established the inherent tethering functions of human Rab-family small GTPases (Tamura and Mima, 2014;Inoshita and Mima, 2017;Mima, 2018;Segawa et al., 2019), following the pioneering work of Lo et al. (2012) which reported for the first time the intrinsic tethering activity of endosomal Ypt/Rab-family proteins in the yeast Saccharomyces cerevisiae. ...
Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.
... The process of membrane tethering is vital for determining the spatiotemporal specificity of intracellular membrane trafficking, before the irreversible final steps of membrane docking and fusion mediated by SNARE-family proteins (Jahn and Scheller, 2006), which are another critical layers to confer the fidelity of membrane trafficking (McNew et al., 2000;Parlati et al., 2002;Izawa et al., 2012;Furukawa and Mima, 2014). A large body of prior studies on membrane tethering or vesicle tethering (or capture) have identified a number of the protein components essential for membrane tethering (Yu and Hughson, 2010;Kuhlee et al., 2015;Cheung and Pfeffer, 2016;Spang, 2016;Witkos and Lowe, 2016;Gillingham and Munro, 2019), which include the Uso1/p115 coiled-coil protein (Sapperstein et al., 1995;Sapperstein et al., 1996;Barlowe, 1997;Cao et al., 1998), golgin-family coiled-coil proteins (Drin et al., 2008;Wong and Munro, 2014;Cheung et al., 2015), the EEA1 coiled-coil protein (Murray et al., 2016), and a diversified set of multisubunit tethering complexes, such as the HOPS complex (Price et al., 2000;Stroupe et al., 2009;Hickey and Wickner, 2010;Ho and Stroupe, 2015;Ho and Stroupe, 2016), the exocyst complex (TerBush et al., 1996;Guo et al., 1999;Rossi et al., 2020), the COG complex (Ungar et al., 2002;Zolov and Lupashin, 2005;Shestakova et al., 2006), the Dsl1 complex (Reilly et al., 2001;Zink et al., 2009;Ren et al., 2009), the GARP complex (Conibear and Stevens, 2000;Perez-Victoria et al., 2008;Perez-Victoria and Bonifacino, 2009), and the TRAPP complex (Sacher et al., 2001;Cai et al., 2005;Cai et al., 2007). It is noteworthy that, in addition to these miscellaneous, sequentially-and structurally-diverse classic tethering factors, our recent reconstitution studies have established the inherent tethering functions of human Rab-family small GTPases (Tamura and Mima, 2014;Inoshita and Mima, 2017;Mima, 2018;Segawa et al., 2019), following the pioneering work of Merz and colleagues, which reported for the first time the intrinsic tethering activity of endosomal Ypt/Rab-family proteins in the yeast Saccharomyces cerevisiae (Lo et al., 2012). ...
Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to drastically enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.
... The Rab subfamily of small GTPases has long been known as regulators of membrane trafficking, and Ypt7p function has been well established at the vacuole (Wichmann et al., 1992) (Haas et al., 1995) (Vollmer et al., 1999) Karim et al., 2018;Lachmann et al., 2012;Langemeyer et al., 2018;Price et al., 2000). Additionally, the Rho subfamily's contributions to the fusion cascade at the yeast author/funder. ...
... https://doi.org/10. 1101/2020 The significant loss of small GTPases Ypt7p and Rho1p from vacuolar membranes (Fig. 3A) is intriguing, as these proteins are known to function in the tethering and docking steps of homotypic fusion Eitzen et al., 2001;Eitzen et al., 2002;Haas et al., 1995;Isgandarova et al., 2007;Jones et al., 2010;Karim et al., 2018;Lachmann et al., 2012;Langemeyer et al., 2018;Logan et al., 2010;Logan et al., 2011;Ohya et al., 1993;Price et al., 2000;Vollmer et al., 1999;Wichmann et al., 1992). N-terminal fusion proteins of GFP-Ypt7p under control of the native promoter , and constitutively expressed DsRed-Rho1p on a centromeric plasmid (this study) were expressed in live yeast. ...
... Fischer)] for one hour to visualize. Primary antibodies utilized for this study were used as previously described (Bodman et al., 2015;Haas et al., 1995;Haas and Wickner, 1996;Mayer and Wickner, 1997;Peng et al., 2006;Price et al., 2000;Seals et al., 2000) Proton pumping ...
The role of sphingolipids in controlling the endolysosomal membrane trafficking remains unclear. Here, we show that in Saccharomyces cerevisiae sphingolipids containing very long-chain fatty-acids (VLCAs) promote homotypic vacuolar fusion. Yeast that lack the C26 VLCA elongase Elo3p display morphological and vacuolar abnormalities. Vacuoles isolated from these cells displayed reduced levels of in vitro fusion, which we traced to a block in tethering and docking. We found that C26 VLCFA deficient yeast mislocalize fusion markers, and the small GTPases Rho1p and Ypt7p fail to selectively concentrate at the boundary and vertex domains of vacuoles isolated from these yeasts. Surprisingly, we only observed mild changes to the localization of other regulatory lipids, but membrane fluidity and solubility was significantly altered. Taken together, these results suggest that sphingolipids containing C26 VLCFAs act as regulatory lipids in the homotypic vacuolar fusion cascade by assembling membrane microdomains that promote the protein and lipid machinery required for the tethering and docking of vacuoles.
Summary
Many sphingolipids contain very-long chain fatty-acids with 26 carbons. The deletion of Elo3, the elongase that adds the final two carbons results in pleiotropic effects that negatively alter membrane fusion at the tethering and docking stages.
