Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6.

SNARE proteins are required for fusion of transport vesicles with target membranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole. Here we identified a previously uncharacterized gene, VTS1, and the R-SNARE YKT6 both as multicopy and as low copy suppressors of the growth and vacuolar transport defect in vti1-2 cells. Ykt6p was known to function in retrograde traffic to the cis-Golgi and homotypic vacuolar fusion. We found that VTI1 and YKT6 also interacted in traffic to the prevacuole and vacuole, indicating that these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p and Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively. As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of specificity. To study the role of the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and ykt6-R165Q. SNARE complexes to which Ykt6p contributed a fourth glutamine residue in the 0 layer were nonfunctional, suggesting an essential function for arginine in the 0 layer of these complexes. vti1-Q158R cells had severe defects in several transport steps, indicating that the second arginine in the 0 layer interfered with function.

A SNARE complex mediating fusion of late endosomes defines conserved properties of SNARE structure and function.

Sets of SNARE proteins mediate membrane fusion by assembling into core complexes. Multiple SNAREs are thought to function in different intracellular trafficking steps but it is often unclear which of the SNAREs cooperate in individual fusion reactions. We report that syntaxin 7, syntaxin 8, vti1b and endobrevin/VAMP-8 form a complex that functions in the fusion of late endosomes. Antibodies specific for each protein coprecipitate the complex, inhibit homotypic fusion of late endosomes in vitro and retard delivery of endocytosed epidermal growth factor to lysosomes. The purified proteins form core complexes with biochemical and biophysical properties remarkably similar to the neuronal core complex, although each of the four proteins carries a transmembrane domain and three have independently folded N-terminal domains. Substitution experiments, sequence and structural comparisons revealed that each protein occupies a unique position in the complex, with syntaxin 7 corresponding to syntaxin 1, and vti1b and syntaxin 8 corresponding to the N- and C-terminal domains of SNAP-25, respectively. We conclude that the structure of core complexes and their molecular mechanism in membrane fusion is highly conserved between distant SNAREs.

The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex.

Specific soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins are required for different membrane transport steps. The SNARE Vti1a has been colocalized with Golgi markers and Vti1b with Golgi and the trans-Golgi network or endosomal markers in fibroblast cell lines. Here we study the distribution of Vti1a and Vti1b in brain. Vti1b was found in synaptic vesicles but was not enriched in this organelle. A brain-specific splice variant of Vti1a was identified that had an insertion of seven amino acid residues next to the putative SNARE-interacting helix. This Vti1a-beta was enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals. Vti1a-beta also copurified with the synaptic vesicle R-SNARE synaptobrevin during immunoisolation of synaptic vesicles and endosomes. Therefore, both synaptobrevin and Vti1a-beta are integral parts of synaptic vesicles throughout their life cycle. Vti1a-beta was part of a SNARE complex in nerve terminals, which bound N-ethylmaleimide-sensitive factor and alpha-SNAP. This SNARE complex was different from the exocytic SNARE complex because Vti1a-beta was not coimmunoprecipitated with syntaxin 1 or SNAP-25. These data suggest that Vti1a-beta does not function in exocytosis but in a separate SNARE complex in a membrane fusion step during recycling or biogenesis of synaptic vesicles.

The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment.

Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.

Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion.

Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.

A new beat for the SNARE drum.

Eukaryotic cells contain membrane-bound compartments that are connected by trafficking of vesicular intermediates. To maintain compartmental organization, proper targeting of transport vesicles is achieved by specific evolutionarily conserved transmembrane proteins that reside on vesicles and target membranes. According to the original SNARE hypothesis, the formation of a complex of an NEM-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs) and membrane-bound SNAP receptor proteins (SNAREs) ensures docking specificity and leads to membrane fusion driven by the ATPase activity of NSF. Recent results have challenged some aspects of this hypothesis and led to a reassessment of models of SNARE interactions and the events leading to vesicle docking and fusion.

The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p.

Membrane traffic in eukaryotic cells requires that specific v-SNAREs on transport vesicles interact with specific t-SNAREs on target membranes. We identified a novel Saccharomyces cerevisiae v-SNARE (Vti1p) encoded by the essential gene, VTI1. Vti1p interacts with the prevacuolar t-SNARE Pep12p to direct Golgi to prevacuolar traffic. vti1-1 mutant cells missorted and secreted the soluble vacuolar hydrolase carboxypeptidase Y (CPY) rapidly and reversibly when vti1-1 cells were shifted to the restrictive temperature. However, overexpression of Pep12p suppressed the CPY secretion defect exhibited by vti1-1 cells at 36 degrees C. Characterization of a second vti1 mutant, vti1-11, revealed that Vti1p also plays a role in membrane traffic at a cis-Golgi stage. vti1-11 mutant cells displayed a growth defect and accumulated the ER and early Golgi forms of both CPY and the secreted protein invertase at the nonpermissive temperature. Overexpression of the yeast cis-Golgi t-SNARE Sed5p suppressed the accumulation of the ER form of CPY but did not lead to CPY transport to the vacuole in vti1-11 cells. Overexpression of Sed5p allowed growth in the absence of Vti1p. In vitro binding and coimmunoprecipitation studies revealed that Vti1p interacts directly with the two t-SNAREs, Sed5p and Pep12p. These data suggest that Vti1p plays a role in cis-Golgi membrane traffic, which is essential for yeast viability, and a nonessential role in the fusion of Golgi-derived vesicles with the prevacuolar compartment. Therefore, a single v-SNARE can interact functionally with two different t-SNAREs in directing membrane traffic in yeast.

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

GTP cleavage by the small GTP-binding protein Rab3A is associated with exocytosis of synaptic vesicles induced by alpha-latrotoxin.

Neurotransmitter release from presynaptic nerve terminals is a highly regulated form of exocytosis. Small GTP-binding proteins of the Rab family have been proposed to act as central regulators in this process that cycle between a GTP- and GDP-bound form. Previous work has shown that the synaptic vesicle protein Rab3A undergoes a membrane association-dissociation cycle that is associated with neurotransmitter release. Using isolated nerve terminals as our model system, we have now analyzed the GDP/GTP status of Rab3A. Synaptic vesicle-bound Rab3A was almost exclusively in the GTP form whereas cytosolic Rab3A contained only GDP. Approximately equal amounts of GTP and GDP were found in the pool of Rab3A localized to a membrane fraction containing plasma membrane-synaptic vesicle complexes. In contrast to Rab3A, Rab5 (an endosomal G-protein) was predominantly GDP-bound in all analyzed compartments. To analyze whether Rab3A-bound GTP is cleaved during exocytosis, synaptosomes were stimulated with alpha-latrotoxin, the active component of black widow spider venom. This resulted in massive exocytosis. A significant increase of the GDP/GTP ratio of Rab3A was observed under these conditions that was not due to a nonspecific loss of high energy nucleotides. Our findings suggest that cleavage of Rab3A-bound GTP is a crucial step in regulated exocytosis of synaptic vesicles.

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis.

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.