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The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include p...
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... elegans and Drosophila melanogaster contain only 12-13 genes encoding proteins with recognizable FYVE domains containing some or all of the structural features necessary to bind PtdIns3P. All of these have homologues in the human genome (Table 1), and nine are expressed in all three organisms. Cladograms Other FYVE-domain containing proteins are conserved among two of these three species; for example, the prototypical FYVE domain-containing protein EEA1 does not appear to have a homologue in D. melanogaster. ...
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Recent work from our laboratory has demonstrated that the phosphoinositide PtdIns(3,5)P2 has an essential role in autophagy in the mammalian nervous system. This low abundance, signaling lipid is synthesized by an enzyme complex that is localized on the vacuole membrane in yeast and in the endosome/lysosome compartment in mammalian cells. In mice w...
The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in ≈30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-β receptor. To develop a further understanding of the biological roles of this protein family,...
Of the seven phosphoinositides, PtdIns5P remains the most enigmatic. However, recent research has begun to elucidate its physiological functions. It is now clear that PtdIns5P is found in several distinct subcellular locations, and the identification of a number of PtdIns5P-binding proteins points to its involvement in a variety of key processes, i...
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... The FYVE domain deletion did not interfere with the RUFY4 interaction with Rab7 and LAMP2 (Fig. 4g, h and Fig. S11d, e). However, the FYVE domain is known to be required for binding to PI(3)P, 22,37 and indeed, its deletion disrupted the association of RUFY4 with PI(3)P-containing organelles in HeLa cells (Fig. 4i). This suggests that the FYVE domain of RUFY4 mediates endo-lysosomal membrane binding. ...
Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.
... Besides the BRX domain recognized for mediating protein-protein interactions 23 , four additional subdomains include a Pleckstrin Homology (PH) domain, a cluster of Regulator of Chromosome Condensation 1 (RCC1) repeats, an FYVE (Fab1, YOTB, Vac1, and EEA1) zinc finger domain, and a Coiled-coil (CC) domain (Fig. 1c). The two phospholipid-binding domains (PH and FYVE) hint possible functional connection of PRAF with phospholipid signaling and membrane trafficking 25,26 . The CC domain could mediate protein-protein interaction and the RCC1 domain was found to perform many functions, such as acting as a Guanine nucleotide exchange factor (GEF) to activate Ran GTPases 27,28 . ...
Cell polarity is a fundamental feature underlying cell morphogenesis and organismal development. In the Arabidopsis stomatal lineage, the polarity protein BASL controls stomatal asymmetric cell division. However, the cellular machinery by which this intrinsic polarity site is established remains unknown. Here, we identify the PRAF/RLD proteins as BASL physical partners and mutating four PRAF members leads to defects in BASL polarization. Members of PRAF proteins are polarized in stomatal lineage cells in a BASL-dependent manner. Developmental defects of the praf mutants phenocopy those of the gnom mutants. GNOM is an activator of the conserved Arf GTPases and plays important roles in membrane trafficking. We further find PRAF physically interacts with GNOM in vitro and in vivo. Thus, we propose that the positive feedback of BASL and PRAF at the plasma membrane and the connected function of PRAF and GNOM in endosomal trafficking establish intrinsic cell polarity in the Arabidopsis stomatal lineage.
... FYVE-domain-bearing proteins (for Fab1, YOTB/ZK632.12, Vac1, and EEA1) are specifically found in association with membranous organelles enriched in PtdIns(3)P and highly conserved among eukaryotes, including yeast (Hayakawa et al., 2007; Figure 2). FYVE domains adopt a zinc finger conformation (Misra and Hurley, 1999;Kutateladze and Overduin, 2001). ...
... considered as an ortholog of human EEA1 shows some sequence similarities with the RUFY family. T10G3.5 exhibits PtdIns(3)P binding activity and is involved in endocytosis, being mostly expressed in epidermis and intestine of C. elegans (Hayakawa et al., 2007). In chordates, Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) and FYVE And Coiled-Coil Domain Autophagy Adaptor 1 (FYCO1), display structural and functional features, potentially categorizing them as RUFY proteins. ...
Intracellular trafficking is essential for cell structure and function. In order to perform key tasks such as phagocytosis, secretion or migration, cells must coordinate their intracellular trafficking, and cytoskeleton dynamics. This relies on certain classes of proteins endowed with specialized and conserved domains that bridge membranes with effector proteins. Of particular interest are proteins capable of interacting with membrane subdomains enriched in specific phosphatidylinositol lipids, tightly regulated by various kinases and phosphatases. Here, we focus on the poorly studied RUFY family of adaptor proteins, characterized by a RUN domain, which interacts with small GTP-binding proteins, and a FYVE domain, involved in the recognition of phosphatidylinositol 3-phosphate. We report recent findings on this protein family that regulates endosomal trafficking, cell migration and upon dysfunction, can lead to severe pathology at the organismal level.
... As examples, the best known mammalian FYVE-domain protein, EEA1, is an effector of the Rab5 small GTPase that mediates tethering of endosomes and concomitant fusion of early endosomes [3]; in mammal and yeast, HRS/Vps27p acts as an ESCRT (Endosomal sorting complex required for transport)-0 component to regulate the endosomal recruitment of other ESCRT modules and the sorting of ubiquitinated membrane proteins [4,5]; mammalian FYCO1 is a Rab7 effector that mediate microtube-directed transport of vesicles and autophagosomes [6,7]. Besides canonical endosomal trafficking, recent studies also demonstrate the critical role of several FYVE domain proteins in autophagosome formation and autophagic degradation (for review, please see [8,9]). ...
The FYVE domain is a double zinc finger-like domain that predominantly binds phosphatidylinositol 3-phosphate. The FYVE domain is usually found in proteins primarily involved in regulating various aspects of endomembrane homeostasis, including endosome tethering, endocytic recycling, membrane protein sorting, and autophagosome maturation. Whereas FYVE domain proteins have been extensively studied in mammals and yeast, only a few FYVE domain proteins have been identified and characterized in plants. Here, by using as an example FREE1 (FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1), a protein previously identified by us as a critical factor for endosomal trafficking, we describe methods to determine its lipid binding properties and endosomal localization. In addition, we also demonstrate a method to quickly test whether an FYVE domain protein is involved in endosomal sorting in plant cells.
... Structurally, it was demonstrated that the isolated FYVE domain of several zfyve members, mainly localize to nucleus and the cytoplasm, but upon dimerization stimulation the FYVE domains shift to the endosomes (Hayakawa et al., 2004;Hayakawa, Hayes, Leonard, Lambright, & Corvera, 2007). This implies that oligomerization favors endosomal localization. ...
Smad Anchor for Receptor Activation (SARA, zfyve9) has been classically observed in early endosomes of different cells types where it regulates vesicular transport of proteins and membrane components. Very few other members of the zinc finger FYVE domain‐containing family (zfyve) have different functions other than controlling membrane trafficking. By analyzing SARA localization throughout mouse embryonic brain development, we detected that besides the endosomal localization it also targets neuronal nuclei, specifically of the cortical layers V/VI. These findings were confirmed in human brain organoids. When evaluating neuronal cell lines, we found that SARA accumulates in nuclei of PC‐12 cells, but not Neuro‐2a; highlighting its specificity. SARA functions as a specific marker of the deep cortical layers until the first postnatal week. This temporal regulation corresponds with the final phases of neuron differentiation, such as soma ventral translocation and axonal targeting. In sum, here we report that SARA localization during brain development is temporarily regulated, and layer specific. This defined pattern helps in the identification of early‐born cortical neurons. We further show that other zfyve family members (FYCO1, WDFY3, Hrs) also distribute to nuclei of different cells in the brain cortex, which raises the possibility that this might be an extended feature within the protein family. This article is protected by copyright. All rights reserved. Smad Anchor for Receptor Activation (SARA), besides localizing to early endosomes, exhibits a distinctive nuclear distribution in deep‐layer neurons of the cortex (layers V and VI). This pattern allows the identification of specific neuronal subpopulations.
... Specific PI-binding domains have been described in eukaryotic proteins (20)(21)(22), some of which specifically bind PI3P or PI4P. The FYVE domain (named after the four proteins harboring the domain: Fab1p, YOTB, Vac1p, and EEA1) is between 60 and 80 residues long, and its PI3P-binding site is formed by the positively charged amino acid residues in the conserved WxxD, (R/K)(R/K)HHCR, and RVC sequence (23)(24)(25). The Phox homology (PX) domain is generally between 110 and 140 residues long, and its consensus binding motif for PI3P binding is R(Y/F)x 23-30 Kx 13-23 R (26,27). ...
... effectors are usually positive or aromatic amino acids. This observation also fits the known information about the eukaryotic PX domain, which binds PI3P and harbors the consensus R(Y/F)x 23-30 Kx 13-23 R sequence (26,27); the eukaryotic FYVE domain, which binds PI3P and harbors the consensus WxxD, (R/K)(R/K)HHCR, and RVC sequence (23)(24)(25); and the PI4P-C domain, present in the Legionella homologous effectors SidC and SdcA, in which arginine, tryptophan, and phenylalanine residues are required for PI4P binding (43). Our second finding is that the residues essential for PI binding in the newly identified PI3P-binding LEDs and the PI4P-M-binding domain are located at or close to the end of an alpha-helix or a beta-strand structure (Fig. 9). ...
Legionella pneumophila and other Legionella species replicate intracellularly using the Icm/Dot type-IV secretion system. In L. pneumophila this system translocates >300 effectors into host cells and in the Legionella genus thousands of effectors were identified, the function of most of which is unknown. Twelve L. pneumophila effectors were previously shown to specifically bind phosphoinositides (PIs) using dedicated domains. We found that PIs-binding domains of effectors are usually not homologous to one another; they are relatively small and located at the effectors C-termini. We used the previously identified Legionella effectors domains (LEDs) with unknown function and the above characteristics of effectors PI-binding domains to discover novel PIs-binding LEDs. We identified three predicted PIs-binding LEDs that are present in 14 L. pneumophila effectors and in >200 effectors in the Legionella genus. Using an in-vitro protein-lipid overlay assay, we found that 11 of these L. pneumophila effectors specifically bind phosphatidylinositol 3-phosphate (PI3P), almost doubling the number of L. pneumophila effectors known to bind PIs. Further, we identified in each of these newly discovered PI3P-binding LEDs conserved, mainly positively charged, amino acids that are essential for PI3P-binding. Our results indicate that Legionella effectors harbor unique domains, shared by many effectors, which directly mediate PI3P-binding.
... This result suggests that PI3K activity is a prerequisite for the VRK2A-dependent enhancement of Akt kinase activity at the lysosomes. We showed that after the induction of autophagy, which increases lysosomal PtdIns(3)P amounts, Akt accumulated via Phafin2 in a manner that depended on PtdIns(3)P interaction [36,80]. ...
Serine–threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt–Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight–mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine–threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2–Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2–Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2–Akt complexes in the lysosomes via modulation of autophagy.
... forms parallel coiled-coil dimer, which bridges endosomes through the FYVE domain and through the interaction with the SNARE protein syntaxin 6 to mediate homotypic endosomal fusion (Figure13) (Mills et al., 1998, Hayakawa et al., 2007. Vesicles fusion: Rab GTPases also regulate SNARE-dependent fusion of transport vesicles to target membranes. ...
Tunneling nanotubes are actin-based cell protrusions that mediate cell-to-cell communication by transferring cellular cargos. The different types of intercellular communication are increasing by being considered as potential targets for the treatment of various diseases, such as infectious diseases linked to viruses and bacteria, cancers or neurodegenerative diseases. Recent studies have highlighted a prion-like mechanism of propagation of protein misfolding in a variety of common, non-infectious, neurodegenerative diseases such as Alzheimer’s disease (AD), Frontotemporal dementia (FTD), Parkinson’s disease (PD), and Polyglutamine (PolyQ) diseases, which are characterized by the accumulation of misfolded proteins in the brain of patients. Thus, new therapeutic strategies to block propagation of protein misfolding throughout the brain can be envisaged. It has been shown that TNTs might play a critical role in spreading of prion aggregates within the CNS and from the periphery. Therefore, the study of mechanism of TNT formation could provide new insights on the mechanism of disease propagation and novel therapeutic targets. The aim of my thesis was to study the role of TNT-mediate protein aggregates transfer between cells and to investigate the mechanism of TNT formation. In our lab, we already reported TNT mediate prion transfer between cells. In the first part of my PhD, I further confirmed that prion aggregates transfer between neuronal CAD cells through TNT inside endocytic vesicles (Zhu et al., 2015). Furthermore in collaboration with a colleague, we provided evidences that prion aggregates could transfer between primary astrocytes and neurons and the transfer was mediated by cell-to-cell contact (Victoria et al., 2016). I also collaborated to another study where we showed that α-synuclein aggregates (Parkinson’s disease) can transfer between cells inside lysosomes, and the intercellular transfer is mediated by TNTs (Abounit et al., 2016).In my second project, in order to investigate the mechanism of TNT formation, I performed a High-content screening of Rab GTPase. I found that Rab8 and Rab11 can promote TNT formation, that Rab8-VAMP3, Rab11-ERM and Rab8-Rab11 cascades are involved in TNT formation. My data suggests that both actin polymerization and membrane trafficking are involved in TNT formation. These results help to shed light on the mechanism of TNT formation, and provide molecular evidences that Rab GTPases regulate this process.
... FYVE domain-containing protein family, belonging to the most ancient and diverse zinc finger superfamily, shares a highly conserved FYVE domain that is characterized by eight cysteine residues responsible for the coordination of two zinc binding in order to stabilize the fold (Stenmark et al. 1996;Burd and Emr 1998). Increasing evidence suggests that FYVE domains mainly play roles in protein-protein or protein-lipid interactions (Shisheva et al. 1999;Hayakawa et al. 2007). In vitro and in vivo experiments demonstrate that the FYVE domain is membrane targeting and specifically binds with PtdIns(3)P, which requires four conserved zinc-binding CxxC motifs and RRHHCR of FYVE domain (Burd and Emr 1998;Gaullier et al. 1998;Tsukazaki et al. 1998). ...
The FYVE domain is a typical zinc finger motif containing four conserved CxxC pairs and has been shown to specifically binds to PtdIns(3)P on the surface of cell membrane. FYVE domain-containing proteins are commonly distributed in eukaryotic cells and have been implicated in diverse functions like signal transduction, membrane trafficking, exocytosis and endocytosis, phosphoinositides (PIs) metabolism, and cytoskeletal regulation. Analysis of the rice genome using comprehensive online databases and research tools resulted in the identification of 19 putative rice FYVE (OsFVYE) proteins. Based on domain architectural and phylogenetic analyses, these OsFYVEs were further classified into six groups. Groups I–V were conserved in Arabidopsis (dicots) and rice (monocot) at both the genetic and protein levels, while group VI was widely present in plants but truncated in Arabidopsis and species of Brassicaceae. Comprehensive and comparative investigation of their expression profiles showed that FYVE genes in plant exhibited a variety of expression patterns during different developmental stages and in response to phytohormones and abiotic stresses. These findings indicated that both OsFYVE and AtFYVE genes may played potential roles in normal plant growth, hormone signal transduction, and abiotic stress tolerance. Results from our study shed light on the potential roles of FYVE proteins in plant growth, development, and stress responses.
... Deletion of vapA leads to increased conidiation and reduced cleistothecia production, and consistently the transcripts of asexual regulators, BrlA and AbaA, are substantially increased in the vapA deletion strain. The VapA protein is localized to the plasma membrane as the other FYVE type zinc finger proteins (Hayakawa et al., 2007). The major function of VapA protein is to recruit the two methyltransferase components of the complex, VipC and VapB, to the plasma membrane and not to release them until an environmental signal triggers their release. ...
Fungal secondary metabolism has become an important research topic with great biomedical and biotechnological value. In the postgenomic era, understanding the diversity and the molecular control of secondary metabolites (SMs) are two challenging tasks addressed by the research community. Discovery of the LaeA methyltransferase 10 years ago opened up a new horizon on the control of SM research when it was found that expression of many SM gene clusters is controlled by LaeA. While the molecular function of LaeA remains an enigma, discovery of the velvet family proteins as interaction partners further extended the role of the LaeA beyond secondary metabolism. The heterotrimeric VelB-VeA-LaeA complex plays important roles in development, sporulation, secondary metabolism, and pathogenicity. Recently, three other methyltransferases have been found to associate with the velvet complex, the LaeA-like methyltransferase F and the methyltransferase heterodimers VipC-VapB. Interaction of VeA with at least four methyltransferase proteins indicates a molecular hub function for VeA that questions: Is there a VeA supercomplex or is VeA part of a highly dynamic cellular control network with many different partners?
