How Tlg2p/syntaxin 16 'snares' Vps45

Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins.     

The Syntaxin Tlg1p Mediates Trafficking of Chitin Synthase III to Polarized Growth Sites in Yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the α factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.     

The Aspergillus nidulans syntaxin PepAPep12 is regulated by two Sec1/Munc‐18 proteins to mediate fusion events at early endosomes,late endosomes and vacuoles

Syntaxins are target‐SNAREs that crucially contribute to determine membrane compartment identity. Three syntaxins, Tlg2p, Pep12p and Vam3p, organize the yeast endovacuolar system. Remarkably, filamentous fungi lack the equivalent of the yeast vacuolar syntaxin Vam3p, making unclear how these organisms regulate vacuole fusion. We show that the nearly essential Aspergillus nidulans syntaxin PepA^Pep12, present in all endocytic compartments between early endosomes and vacuoles, shares features of Vam3p and Pep12p, and is capable of forming compositional equivalents of all known yeast endovacuolar SNARE bundles including that formed by yeast Vam3p for vacuolar fusion. Our data further indicate that regulation by two Sec1/Munc‐18 proteins, Vps45 in early endosomes and Vps33 in early and late endosomes/vacuoles contributes to the wide domain of PepA^Pep12 action. The syntaxin TlgB^Tlg2 localizing to the TGN appears to mediate retrograde traffic connecting post‐Golgi (sorting) endosomes with the TGN. TlgB^Tlg2 is dispensable for growth but becomes essential if the early Golgi syntaxin SedV^Sed5 is compromised, showing that the Golgi can function with a single syntaxin, SedV^Sed5. Remarkably, its pattern of associations with endosomal SNAREs is consistent with SedV^Sed5 playing roles in retrograde pathway(s) connecting endocytic compartments downstream of the post‐Golgi endosome with the Golgi, besides more conventional intra‐Golgi roles.     

Cellular levels of the syntaxin Tlg2p are regulated by a single mode of binding to Vps45p

Sec1p/Munc18 (SM) proteins play a key role in the regulation of soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptor (SNARE)-mediated intracellular membrane trafficking events in all eukaryotic cells. Understanding the molecular mechanisms by which SM proteins function has not been straight forward as SM proteins bind to their cognate SNARE proteins by at least two distinct mechanisms, suggesting that they provide more than one function. We have previously characterised two binding modes used by the yeast SM protein Vps45p to interact with its SNARE proteins. In one of these modes, the N terminus of the syntaxin Tlg2p inserts into a hydrophobic pocket in the SM protein. We now report that disruption of this high-affinity binding between Vps45p and Tlg2p leads to downregulation of Tlg2p, and propose that this pocket-mode of binding of SM proteins to their cognate syntaxins serves to regulate cellular levels of the syntaxin.     

Concerted auto-regulation in yeast endosomal t-SNAREs

In yeast, the assembly of the target (t)-SNAREs [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p] with the vesicular (v)-SNARE Snc2p promotes endocytic fusion. Here, selected mutations and truncations of SNARE proteins were tested in an in vitro fusion assay to identify potential regulatory regions in these proteins, and two distinct regions were found. The first is represented by the combined effect of the three t-SNARE N-terminal regions and the second is located within the Tlg1p SNARE motif. These internal controls provide a potential mechanism to enable SNARE-dependent fusion to be regulated.     

The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar protein transport. Our screen for temperature-sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non-permissive temperature. Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain. Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion. In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step. Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes.     

Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome-Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.     

Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.     

Pep12p is a multifunctional yeast syntaxin that controls entry of biosynthetic, endocytic and retrograde traffic into the prevacuolar compartment

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p. Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole. Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC. The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC. Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC. Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27 -independent fashion.     

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6 delta C overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes.     

Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae

Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3(ts) mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.     

A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes.By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.     

Promiscuity in Rab-SNARE interactions

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.     

Structures of yeast vesicle trafficking proteins

In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.     

The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p-Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle.     

Syntaxin 16 is enriched in neuronal dendrites and may have a role in neurite outgrowth

Polarized membrane traffic to different domains of the neuron is well documented, and is required for both establishment and maintenance of neuronal polarity. Some soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, particularly syntaxin 12/13 and TI-VAMP/VAMP7, have known roles in the neuron. We report here that the brain-enriched SNARE syntaxin 16 (Syn 16) is specifically enriched in neuronal dendrites and found at Golgi outposts, thus confirming that Golgi outposts are endowed with a trans-Golgi network (TGN) component. Over-expression of wild type syntaxin 16 moderately stimulates, whereas that of an N-terminal deletion mutant (Syn 16-DeltaNt) inhibits, neurite outgrowth in both mouse Neuro-2a cells and primary cortical neurons. Consistent with an inhibited neurite growth, cells over-expressing Syn 16-DeltaNt have diminished betaIII-tubulin and F-actin labeling. RNA interference-mediated silencing of syntaxin 16 in primary cortical neurons significantly retards neurite outgrowth. Syntaxin 16 may thus play a role in neurite outgrowth and perhaps other specific dendritic anterograde/retrograde traffic.     

Modulation of the K(v)4.3 channel by syntaxin 1A

The SNARE protein syntaxin 1A (Syn1A) is known to inhibit delayed rectifier K(+) channels of the K(v)1 and K(v)2 families with heterogeneous effects on their gating properties. In this study, we explored whether Syn1A could directly modulate K(v)4.3, a rapidly inactivating K(v) channel with important roles in neuroendocrine cells and cardiac myocytes. Immunoprecipitation studies in HEK293 cells coexpressing Syn1A and K(v)4.3 revealed a direct interaction with increased trafficking to the plasma membrane without a change in channel synthesis. Paradoxically, Syn1A inhibited K(v)4.3 current density. In particular, Syn1A produced a left-shift in steady-state inactivation of K(v)4.3 without affecting either voltage dependence of activation or gating kinetics, a pattern distinct from other K(v) channels. Combined with our previous reports, our results further verify the notion that the mechanisms involved in Syn1A-K(v) interactions vary significantly between K(v) channels, thus providing a wide scope for Syn1A modulation of exocytosis and membrane excitability.     

Two syntaxin homologues in the TGN/endosomal system of yeast.

Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v-SNAREs) and target organelles (t-SNAREs) that bind to each other and mediate bilayer fusion. All known SNARE-mediated fusion events involve a member of the syntaxin family of t-SNAREs. Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized. We describe here the remaining two, Tlg1p and Tlg2p. These have the expected biochemical properties of t-SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively. They co-precipitate with the v-SNARE Vti1p, which is implicated in Golgi-endosome traffic and, remarkably, binds to five different syntaxins. Tlg1p also binds the plasma membrane v-SNARE Snc1p. Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins. However, neither is required for intra-Golgi traffic. Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p.