Characterization of the human GARP (Golgi associated retrograde protein) complex

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.     

Direct interaction between the COG complex and the SM protein,Sly1, is required for Golgi SNARE pairing

The crucial roles of Sec1/Munc18 (SM)‐like proteins in membrane fusion have been evidenced in genetic and biochemical studies. SM proteins interact directly with SNAREs and contribute to SNARE pairing by a yet unclear mechanism. Here, we show that the SM protein, Sly1, interacts directly with the conserved oligomeric Golgi (COG) tethering complex. The Sly1–COG interaction is mediated by the Cog4 subunit, which also interacts with Syntaxin 5 through a different binding site. We provide evidence that disruption of Cog4–Sly1 interaction impairs pairing of SNAREs involved in intra‐Golgi transport thereby markedly attenuating Golgi‐to‐ER retrograde transport. These results highlight the mechanism by which SM proteins link tethering to SNAREpin assembly.     

Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5a/Sed5 enhances intra-Golgi SNARE complex stability

Tethering factors mediate initial interaction of transport vesicles with target membranes. Soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) enable consequent docking and membrane fusion. We demonstrate that the vesicle tether conserved oligomeric Golgi (COG) complex colocalizes and coimmunoprecipitates with intra-Golgi SNARE molecules. In yeast cells, the COG complex preferentially interacts with the SNARE complexes containing yeast Golgi target (t)-SNARE Sed5p. In mammalian cells, hCog4p and hCog6p interact with Syntaxin5a, the mammalian homologue of Sed5p. Moreover, fluorescence resonance energy transfer reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady-state levels of the intra-Golgi SNARE complex. Finally, overexpression of the hCog4p N-terminal Syntaxin5a-binding domain destabilizes intra-Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t-SNARE Syntaxin5a/Sed5p and enhancing the stability of intra-Golgi SNARE complexes.     

A Highlights from MBoC Selection: Ang2/Fat-Free Is a Conserved Subunit of the Golgi-associated Retrograde Protein Complex

The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.     

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in b16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.     

The Habc domain and the SNARE core complex are connected by a highly flexible linker

Syntaxin 1a is a member of the SNARE superfamily of small, mostly membrane-bound proteins that mediate membrane fusion in all eukaryotic cells. Upon membrane fusion, syntaxin 1 forms a stable complex with its partner SNAREs. Syntaxin contains a C-terminal transmembrane domain, an adjacent SNARE motif that interacts with its partner SNAREs, and an N-terminal Habc domain. The Habc domain reversibly folds back upon the SNARE motif, resulting in a "closed" conformation that is stabilized by binding to the protein munc18. The SNARE motif and the Habc domain are separated by a linker region of about 40 amino acids. When syntaxin is complexed with munc18, the linker is structured and consists of a mix of turns and small alpha-helices. When syntaxin is complexed with its partner SNAREs, the Habc domain is dissociated, but the structure of the linker region is not known. Here we used site-directed spin labeling and EPR spectroscopy to determine the structure of the linker region of syntaxin in the SNARE complex. We found that the entire linker region of syntaxin is unstructured except for three residues at the N-terminal and six residues at the C-terminal boundary whereas the structures of the flanking regions in the Habc domain and the SNARE motif correspond to the high-resolution structures of the isolated fragments. We conclude that the linker region exhibits a high degree of conformational flexibility.     

α‐Synuclein sequesters arachidonic acid to modulate SNARE‐mediated exocytosis

α‐Synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. α‐Synuclein has been proposed to regulate soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, α‐synuclein fails to interact with SNARE proteins in conventional protein‐binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both α‐synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by α‐synuclein, both in vitro and in vivo. α‐Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of α‐synuclein in the modulation of neurotransmission.     

Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.     

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

Sequential involvement of p115, SNAREs, and Rab proteins in intra-Golgi protein transport

Delivery of transport vesicles to their receptor compartment involves tethering, priming, and fusion. Soluble NSF attachment protein-alpha (alphaSNAP) mediates the disruption of SNAREs by N-ethylmaleimide sensitive factor (NSF) and was employed to determine the hierarchy of proteins responsible for intra-Golgi protein transport. The N-terminal 23 amino acids of alphaSNAP are necessary for SNARE binding. The antibody 2F10 recognizes this SNARE interaction domain of alphaSNAP and inhibits intra-Golgi protein transport reversibly. This antibody was applied to modify the transport assay to determine the protein requirements relative to the action of alphaSNAP and NSF. We found that 1) p115 acts independently of alphaSNAP and NSF, 2) SNAREs are required after tethering and interact selectively after activation by alphaSNAP and NSF, and 3) Rab proteins act after SNARE activation and before fusion.     

The COG complex interacts directly with Syntaxin 6 and positively regulates endosome-to-TGN retrograde transport

The conserved oligomeric Golgi (COG) complex has been implicated in the regulation of endosome to trans-Golgi network (TGN) retrograde trafficking in both yeast and mammals. However, the exact mechanisms by which it regulates this transport route remain largely unknown. In this paper, we show that COG interacts directly with the target membrane SNARE (t-SNARE) Syntaxin 6 via the Cog6 subunit. In Cog6-depleted cells, the steady-state level of Syntaxin 6 was markedly reduced, and concomitantly, endosome-to-TGN retrograde traffic was significantly attenuated. Cog6 knockdown also affected the steady-state levels and/or subcellular distributions of Syntaxin 16, Vti1a, and VAMP4 and impaired the assembly of the Syntaxin 6-Syntaxin16-Vti1a-VAMP4 SNARE complex. Remarkably, overexpression of VAMP4, but not of Syntaxin 6, bypassed the requirement for COG and restored endosome-to-TGN trafficking in Cog6-depleted cells. These results suggest that COG directly interacts with specific t-SNAREs and positively regulates SNARE complex assembly, thereby affecting their associated trafficking steps.     

COG6 Interacts with a Subset of the Golgi SNAREs and Is Important for the Golgi Complex Integrity

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The conserved oligomeric Golgi (COG) complex has been specifically implicated in the tethering of retrograde intra‐Golgi vesicles. Using yeast two‐hybrid and co‐immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27 and SNAP29. Interaction with SNAREs is accomplished via the universal SNARE‐binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE‐binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6–SNARE interactions are important for both COG6 localization and Golgi integrity .     

The Caenorhabditis elegans GARP complex contains the conserved Vps51 subunit and is required to maintain lysosomal morphology

In yeast the Golgi-associated retrograde protein (GARP) complex is required for tethering of endosome-derived transport vesicles to the late Golgi. It consists of four subunits--Vps51p, Vps52p, Vps53p, and Vps54p--and shares similarities with other multimeric tethering complexes, such as the conserved oligomeric Golgi (COG) and the exocyst complex. Here we report the functional characterization of the GARP complex in the nematode Caenorhabditis elegans. Furthermore, we identified the C. elegans Vps51 subunit, which is conserved in all eukaryotes. GARP mutants are viable but show lysosomal defects. We show that GARP subunits bind specific sets of Golgi SNAREs within the yeast two-hybrid system. This suggests that the C. elegans GARP complex also facilitates tethering as well as SNARE complex assembly at the Golgi. The GARP and COG tethering complexes may have overlapping functions for retrograde endosome-to-Golgi retrieval, since loss of both complexes leads to a synthetic lethal phenotype.     

Syntaxin 16: Unraveling cellular physiology through a ubiquitous SNARE molecule

Syntaxin 16 (Syx16) is member of the soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) family of molecules that functions in membrane fusion in eukaryotic cells. A rather ubiquitously expressed, tail‐anchored membrane protein localized mainly at the trans‐Golgi network (TGN), it mediates primarily retrograde endosomal‐TGN transport. In spite of its ubiquitous expression, Syx16 has specific and interesting roles in the physiology of specialized cells, including Glut4 dynamics, dendritic outgrowth‐related membrane traffic, and cytokinesis. We discussed these physiological functions of Syx16 in the light of what is known of its subcellular localization, vesicular trafficking pathways involved, cognate SNARE partners and other interacting proteins. Further, we speculate on some possible pathophysiological roles of Syx16. J. Cell. Physiol. 225: 326–332, 2010. © 2010 Wiley‐Liss, Inc.     

Interaction of Golgin‐84 with the COG Complex Mediates the Intra‐Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.     

The cystic fibrosis transmembrane conductance regulator's expanding SNARE interactome

The cystic fibrosis transmembrane conductance regulator (CFTR) interacts with multiple N-ethylmaleimide sensitive factor attachment protein (SNARE) molecules largely via its N-terminal cytoplasmic domain. The earliest known among these SNAREs are the cognate Q-SNARE pair of Syntaxin 1A (STX1A) and SNAP23 on the plasma membrane. These SNAREs affect CFTR chloride channel gating. CFTR exocytosis/recycling in intestinal epithelial cells is dependent on another SNARE located in the apical plasma membrane, STX3. Members of the STX8/STX7/vesicle transport through interaction with t-SNAREs homolog 1b/VAMP8 SNARE complex, which function in early to late endosome/lysosome traffic, are all known to interact with CFTR. Two SNAREs, STX6 and STX16 that function at the trans-Golgi network (TGN), have now been revealed as members of the CFTR SNARE interactome. We summarize here the SNAREs that interact with CFTR and discuss the roles of these SNAREs in the intracellular trafficking of CFTR and CFTR-associated pathophysiology.     

Trans-Golgi network syntaxin 10 functions distinctly from syntaxins 6 and 16

Syntaxin 10 is a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein localized to the trans-Golgi network (TGN), where two other members of the syntaxin family, syntaxins 6 and 16, also reside. The role of syntaxin 10 in regulating TGN protein traffic is not yet defined. Syntaxin 10 co-localizes well with syntaxins 6 and 16 at the TGN in interphase cells, and appears to interact with both syntaxin 6 and 16 as evidenced by co-immunoprecipitation analyses. However, unlike syntaxin 6 and 16, neither syntaxin 10 antibodies nor its cytosolic domain inhibits endosome-TGN transport of shiga toxin. Syntaxin 16 knockdown with small interfering RNA (siRNA) affects the TGN localization of syntaxin 6 but not syntaxin 10, and clearly inhibits endosome-TGN transport. On the other hand, knockdown of syntaxin 10 expressions had no significant effect on the TGN localization of syntaxin 6 and 16, and did not inhibit endosome-TGN transport. Unlike syntaxin 16, syntaxin 10 does not interact specifically with Vps45, the Sec1/Munc18 (SM) family member at the TGN. On the other hand, syntaxin 10 reciprocally co-immunoprecipitated endosomal syntaxin 12/13, and knockdown of syntaxin 10 expressions affects the surface expression of transferrin receptor (TfR) and seems to induce the formation of an immobile TfR pool. Therefore, in spite of its co-localization and possible interaction with syntaxin 16, syntaxin 10 is not part of the syntaxin 16-based SNARE complex involved in endosome-TGN transport, and may have a hitherto unrecognized function in the TGN-endosome boundary.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Syntaxin 6: the promiscuous behaviour of a SNARE protein

Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.     

Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.