ATP-independent control of Vac8 palmitoylation by a SNARE subcomplex on yeast vacuoles

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation.     

The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion

The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes during priming. Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified. Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion. This subreaction is controlled by a Sec17-independent function of Sec18. Our data indicate that Ykt6 presents Pal-CoA via its N-terminal longin domain to Vac8, while transfer to Vac8's SH4 domain occurs spontaneously and not enzymatically. The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events.     

Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole

Intracellular membrane fusion requires that membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins on both vesicle and target membranes form a highly specific complex necessary to bring the membranes close in space. Ykt6p is a yeast R-SNARE protein that has been implicated in retrograde transport to the cis-Golgi compartment. Ykt6p has been also been found to fractionate with vacuole membranes and participate in a vacuolar SNARE complex in homotypic vacuole fusion. To investigate the role of Ykt6p in membrane traffic to the vacuole we generated temperature-sensitive mutations in YKT6. One mutation produces an early Golgi block to secretion, and overexpression of the SNARE protein Sft1p suppresses the growth and secretion defects of this mutation. These results are consistent with Ykt6p and Sft1p participating in a SNARE complex associated with retrograde transport to the cis-Golgi. A second set of mutations in YKT6 specifically affects post-Golgi membrane traffic to the vacuole, and the effects of these mutations are not suppressed by Sft1p overexpression. Defects are seen in carboxypeptidase Y sorting, alkaline phosphatase transport, and aminopeptidase I delivery, and in one mutant, overexpression of the SNARE protein Nyv1p suppresses the alkaline phosphatase transport defect. By mutationally separating early and late requirements for Ykt6p, our findings have revealed that Ykt6p is a R-SNARE protein that functions directly in the three biosynthetic pathways to the vacuole.     

A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion

Membrane fusion requires priming, the disassembly of cis-SNARE complexes by the ATP-driven chaperones Sec18/17p. Yeast vacuole priming releases Vam7p, a soluble SNARE. Vam7p reassociation during docking allows trans-SNARE pairing and fusion. We now report that recombinant Vam7p (rVam7p) enters into complex with other SNAREs in vitro and bypasses the need for Sec17p, Sec18p, and ATP. Thus, the sole essential function of vacuole priming in vitro is the release of Vam7p from cis-SNARE complexes. In 'bypass fusion', without ATP but with added rVam7p, there are sufficient unpaired vacuolar SNAREs Vam3p, Vti1p, and Nyv1p to interact with Vam7p and support fusion. However, active SNARE proteins are not sufficient for bypass fusion. rVam7p does not bypass requirements for Rho GTPases,Vps33p, Vps39p, Vps41p, calmodulin, specific lipids, or Vph1p, a subunit of the V-ATPase. With excess rVam7p, reduced levels of PI(3)P or functional Ypt7p suffice for bypass fusion. High concentrations of rVam7p allow the R-SNARE Ykt6p to substitute for Nyv1p for fusion; this functional redundancy among vacuole SNAREs may explain why nyv1delta strains lack the vacuole fragmentation seen with mutants in other fusion catalysts.     

Functional analysis of conserved structural elements in yeast syntaxin Vam3p

Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.     

Trans-SNARE interactions elicit Ca2+ efflux from the yeast vacuole lumen

Ca2+ transients trigger many SNARE-dependent membrane fusion events. The homotypic fusion of yeast vacuoles occurs after a release of lumenal Ca2+. Here, we show that trans-SNARE interactions promote the release of Ca2+ from the vacuole lumen. Ypt7p-GTP, the Sec1p/Munc18-protein Vps33p, and Rho GTPases, all of which function during docking, are required for Ca2+ release. Inhibitors of SNARE function prevent Ca2+ release. Recombinant Vam7p, a soluble Q-SNARE, stimulates Ca2+ release. Vacuoles lacking either of two complementary SNAREs, Vam3p or Nyv1p, fail to release Ca2+ upon tethering. Mixing these two vacuole populations together allows Vam3p and Nyv1p to interact in trans and rescues Ca2+ release. Sec17/18p promote sustained Ca2+ release by recycling SNAREs (and perhaps other limiting factors), but are not required at the release step itself. We conclude that trans-SNARE assembly events during docking promote Ca2+ release from the vacuole lumen.     

The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion

The farnesylated SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport.     

Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion

Intracellular membrane fusion requires SNARE proteins in a trans-complex, anchored to apposed membranes. Proteoliposome studies have suggested that SNAREs drive fusion by stressing the lipid bilayer via their transmembrane domains (TMDs), and that SNARE complexes require a TMD in each docked membrane to promote fusion. Yeast vacuole fusion is believed to require three Q-SNAREs from one vacuole and the R-SNARE Nyv1p from its fusion partner. In accord with this model, we find that fusion is abolished when the TMD of Nyv1p is replaced by lipid anchors, even though lipid-anchored Nyv1p assembles into trans-SNARE complexes. However, normal fusion is restored by the addition of both Sec18p and the soluble SNARE Vam7p. In restoring fusion, Sec18p promotes the disassembly of trans-SNARE complexes, and Vam7p enhances their assembly. Thus, either the TMD of this R-SNARE is not essential for fusion, and TMD-mediated membrane stress is not the only mode of trans-SNARE complex action, or these SNAREs have more flexibility than heretofore appreciated to form alternate functional complexes that violate the 3Q:1R rule.     

Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators

Longins are the only R-SNAREs that are common to all eukaryotes and are characterized by a conserved N-terminal domain with a profilin-like fold called a longin domain (LD). These domains seem to be essential for regulating membrane trafficking and they mediate unexpected biochemical functions via a range of protein-protein and intramolecular binding specificities. In addition to the longins, proteins involved in the regulation of intracellular trafficking, such as subunits of the adaptor and transport protein particle complexes, also have LD-like folds. The functions and cellular localization of longins are regulated at several levels and the longin prototypes TI-VAMP, Sec22 and Ykt6 show different distributions among eukaryotes, reflecting their modular and functional diversity. In mammals, TI-VAMP and Ykt6 are crucial for neuronal function, and defects in longin structure or function might underlie some human neurological pathologies.     

Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion.

The vacuole v-t-SNARE complex is disassembled by Sec17p/alpha-SNAP and Sec18p/NSF prior to vacuole docking and fusion. We now report a functional characterization of the vacuolar SNARE Vam7p, a SNAP-25 homolog. Although Vam7p has no hydrophobic domains, it is tightly associated with the vacuolar membrane. Vam7p is a constituent of the vacuole SNARE complex and is released from this complex by the Sec17p/Sec18p/ATP-mediated priming of the vacuoles. Even in the absence of the vacuolar v-SNARE Nyv1p, a subcomplex which includes Vam7p and the t-SNARE Vam3p is preserved. Vam7p is necessary for the stability of the vacuolar SNARE complex, since vacuoles from mutants deleted in VAM7 do not have a Vam3p-Nyv1p complex. Furthermore, Vam7p alone, in the absence of Nyv1p and Vam3p, cannot mediate fusion with wild-type vacuoles, whereas vacuoles with only Nyv1p or Vam3p alone can fuse with wild-type vacuoles in the absence of the other two SNAREs. Thus, Vam7p is important for the stable assembly and efficient function of the vacuolar SNARE complex and maintenance of the vacuolar morphology. This functional characterization of Vam7p suggests a general role for SNAP-25 homologs, not only on the plasma membrane but along the secretory pathway.     

Farnesylation of the SNARE protein Ykt6 increases its stability and helical folding

The evolutionarily conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. While most SNAREs are permanently anchored to membranes by their transmembrane domains, the vesicle-associated SNARE Ykt6 has been found both in soluble and in membrane-bound pools. The R-SNARE Ykt6 is thought to mediate interactions between various Q-SNAREs by a reversible membrane-targeting cycle. Membrane attachment of Ykt6 is achieved by its C-terminal prenylation and palmitoylation motif succeeding the SNARE motif. In this study, we have analyzed full-length farnesylated Ykt6 from yeast and humans by biochemical and structural means. In vitro farnesylation of the C-terminal CAAX box of recombinant full-length Ykt6 resulted in stabilization of the native protein and a more compactly folded structure, as shown by size exclusion chromatography and limited proteolysis. Circular dichroism spectroscopy indicated a specific increase in the helical content of the farnesylated Ykt6 compared to the nonlipidated form or the single-longin domain, which correlated with a marked increase in stability as observed by heat denaturation experiments. Although highly soluble, farnesylated Ykt6 is capable of lipid membrane binding independent of the membrane charge, as shown by surface plasmon resonance. The crystal structure of the N-terminal longin domain of yeast Ykt6 (1-140) was determined at 2.5 Å resolution. As similarly found in a previous NMR structure, the Ykt6 longin domain contains a hydrophobic patch at its surface that may accommodate the lipid moiety. In the crystal structure, this hydrophobic surface is buried in a crystallographic homomeric dimer interface. Together, these observations support a previously suggested closed conformation of cytosolic Ykt6, where the C-terminal farnesyl moiety folds onto a hydrophobic groove in the N-terminal longin domain.     

Identification of functionally interacting SNAREs by using complementary substitutions in the conserved '0' layer

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes form bundles of four parallel alpha-helices. The central '0' layer of interacting amino acid side chains is highly conserved and contains one arginine and three glutamines, leading to the classification of SNAREs into R, Qa, Qb, and Qc-SNAREs. Replacing one of the glutamines with arginine in the yeast exocytotic SNARE complex is either lethal or causes a conditional growth defect that is compensated by replacing the R-SNARE arginine with glutamine. Using the yeast SNARE complex mediating traffic from the endoplasmic reticulum to the Golgi apparatus, we now show that functionally interacting SNAREs can be mapped by systematically exchanging glutamines and arginines in the '0' layer. The Q-->R replacement in the Qb-SNARE Bos1p has the strongest effect and can be alleviated by an Q-->R replacement in the R-SNARE Sec22p. Four Q residues in the central layer caused growth defects above 30 degrees C that were rescued by Q-->R substitutions in the Qa and Qc SNAREs Sed5p and Bet1p, respectively. The sec22(Q)/sed5(R) mutant is temperature sensitive and is rescued by a compensating R-->Q replacement in the R-SNARE Ykt6p. This rescue is attributed to the involvement of Sed5p and Ykt6p in a different SNARE complex that functions in intra-Golgi trafficking.     

Longin-like folds identified in CHiPS and DUF254 proteins: vesicle trafficking complexes conserved in eukaryotic evolution

Eukaryotic protein trafficking pathways require specific transfer of cargo vesicles to different target organelles. A number of vesicle trafficking and membrane fusion components participate in this process, including various tethering factor complexes that interact with small GTPases prior to SNARE-mediated vesicle fusion. In Saccharomyces cerevisiae a protein complex of Mon1 and Ccz1 functions with the small GTPase Ypt7 to mediate vesicle trafficking to the vacuole. Mon1 belongs to DUF254 found in a diverse range of eukaryotic genomes, while Ccz1 includes a CHiPS domain that is also present in a known human protein trafficking disorder gene (HPS-4). The present work identifies the CHiPS domain and a sequence region from another trafficking disorder gene (HPS-1) as homologs of an N-terminal domain from DUF254. This link establishes the evolutionary conservation of a protein complex (HPS-1/HPS-4) that functions similarly to Mon1/Ccz1 in vesicle trafficking to lysosome-related organelles of diverse eukaryotic species. Furthermore, the newly identified DUF254 domain is a distant homolog of the mu-adaptin longin domain found in clathrin adapter protein (AP) complexes of known structure that function to localize cargo protein to specific organelles. In support of this fold assignment, known longin domains such as the AP complex sigma-adaptin, the synaptobrevin N-terminal domains sec22 and Ykt6, and the srx domain of the signal recognition particle receptor also regulate vesicle trafficking pathways by mediating SNARE fusion, recognizing specialized compartments, and interacting with small GTPases that resemble Ypt7.     

Depalmitoylation of Ykt6 prevents its entry into the multivesicular body pathway

The dually lipidated SNARE Ykt6 is found on intracellular membranes and in the cytosol. In this study, we show that Ykt6 localizes to the Golgi as well as endosomal and vacuolar membranes in vivo . The ability of Ykt6 to cycle between the cytosol and the membranes depends on the intramolecular interaction of the N-terminal longin and C-terminal SNARE domains and not on either domain alone. A mutant deficient in this interaction accumulates on membranes and – in contrast to the wild-type protein – does not get released from vacuoles. Our data also indicate that Ykt6 is a substrate of the DHHC (Asp-His-His-Cys) acyltransferase network. Overexpression of the vacuolar acyltransferase Pfa3 drives the F42S mutant not only to the vacuole but also into the vacuolar lumen. Thus, depalmitoylation and release of Ykt6 are needed for its recycling and to circumvent its entry into the endosomal multivesicular body pathway.     

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.     

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.     

Polar transmembrane domains target proteins to the interior of the yeast vacuole

Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly. Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes. However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal. In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum. The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p. It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole. These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.     

Stringent 3Q.1R composition of the SNARE 0-layer can be bypassed for fusion by compensatory SNARE mutation or by lipid bilayer modification

SNARE proteins form bundles of four alpha-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q.1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7(Q283R), yet Vam7(Q283R) is preferred by vacuoles with Nyv1(R191Q). Rotation of the position of the arginine in the 0-layer increases the K(m) for Vam7p but does not affect the maximal rate of fusion. Vam7(Q283R) forms stable 2Q.2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion.     

Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion

Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.