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.     

HOPS prevents the disassembly of trans‐SNARE complexes by Sec17p/Sec18p during membrane fusion

SNARE‐dependent membrane fusion requires the disassembly of cis‐SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans‐SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans‐SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans‐SNARE complex, like cis‐SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans‐, but not in the cis‐, configuration. This selective HOPS preservation of trans‐SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X‐100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans‐SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic.     

Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P₂ ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P₂ , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1 Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1 ; however, deleting both genes did not rescue the pah1 Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity.     

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly,allowing small headgroup lipids to support the terminal steps of membrane fusion

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.     

A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.     

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones

The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.     

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.     

The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP‐25

The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE‐mediated membrane fusion. Recently, a new protein–protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature‐sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18‐1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP‐25b, respectively. Incubation of SNAP‐25b with the Munc18‐1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild‐type Munc18‐1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p–SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP‐25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.      

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.     

Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion

Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones.Biological membrane fusion is the regulated rearrangement of the lipids in two apposed sealed membranes to form one bilayer while mixing lumenal contents without leakage or lysis. It is fundamental for intracellular vesicular traffic, cell growth and division, regulated secretion of hormones and other blood proteins, and neurotransmission and thus has attracted wide and sustained study (1, 2). Its fundamental mechanisms are conserved and employ a Rab-family GTPase, proteins which bind to the GTP-bound form of a Rab, termed its “effectors” (3), and SNARE³ (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (4) with their attendant chaperones. SNAREs are integral or peripheral membrane proteins with characteristic heptad-repeat domains, which can associate in 4-helical coiled-coils (5), termed “cis-SNARE complexes,” if they are all anchored to the same membrane bilayer, or “trans-SNARE complexes” if they are anchored to apposed membranes.Stable membrane proximity (docking) does not suffice for fusion. Studies in model systems have shown that fusion can be promoted by any of several agents, which promote bilayer rearrangement, such as diacylglycerol (6), high levels of calcium (7), viral-encoded fusion proteins (8, 9), or SNAREs (10, 11). These studies frequently employed liposomes or proteoliposomes of simple lipid composition, suggesting that fusion may not have stringent requirements of lipid head group species. However, each of these model fusion reactions is accompanied by substantial lysis (12–15), whereas the preservation of subcellular compartments is a hallmark of physiological membrane fusion.We have studied membrane fusion with the vacuole (lysosome) of Saccharomyces cerevisiae (reviewed in Ref. 16). The fusion of isolated vacuoles requires the Rab Ypt7p, 4 SNAREs (Vam3p, Vti1p, Vam7p, and Nyv1p), the SNARE chaperones Sec17p (α-soluble N-ethylmaleimide-sensitive factor attachment protein)/Sec18p (N-ethylmaleimide-sensitive factor) and the hexameric HOPS complex (17), and key “regulatory” lipids including ERG, phosphoinositides, and DAG (18). HOPS interacts physically or functionally with each component of this fusion system. HOPS stably associates with Ypt7p in its GTP-bound state (19). One HOPS subunit, Vps33p, is a member of the Sec1-Munc18 family of SNARE-binding proteins, and HOPS exhibits direct affinity for SNAREs (17, 20–22) and proofreads correct vacuolar SNARE pairing (23). HOPS also has direct affinity for phosphoinositides (17). The SNAREs on isolated vacuoles are in cis-complexes, which are disassembled by Sec17p, Sec18p, and ATP (24). Docking requires Ypt7p (25) and HOPS (17). During docking, vacuoles are drawn against each other until each has a substantial membrane domain tightly apposed to the other. Each of the proteins (26) and lipids (18) required for fusion becomes enriched in a ring-shaped microdomain, the “vertex ring,” which surrounds the two tightly apposed membrane domains. Not only do the proteins depend on each other, in a cascade fashion, for vertex ring enrichment, and the lipids depend on each other for their vertex ring enrichment as well, but the lipids and proteins are mutually interdependent for their enrichment at this ring-shaped microdomain (18, 27). Fusion occurs around the ring, joining the two organelles. The fusion of vacuoles bearing physiological fusion constituents does not cause measurable organelle lysis, although fusion supported exclusively by higher levels of SNARE proteins is accompanied by massive lysis (28), in accord with model liposome studies (14). Thus fusion microdomain assembly and the coordinate action of SNAREs with other proteins and lipids to promote fusion without lysis are central topics in membrane fusion studies.Reconstitution of fusion with pure components allows chemical definition of essential elements of this biologically important reaction. Although SNAREs can drive a slow fusion of PC/PS proteoliposomes (29), this was not stimulated by HOPS and Sec17p/Sec18p (30). SNARE proteoliposomes bearing all the vacuolar lipids (18, 31–33), PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), showed rapid and efficient fusion that was fully dependent on Sec17p/Sec18p and HOPS (30). The omission of either DAG, ERG, or phosphoinositide from the liposomes caused a marked reduction in fusion (30). We now report that PE and PA are also necessary for rapid and efficient fusion, function in distinct manners, and are required for efficient assembly of newly formed SNARE complexes by the SNARE chaperones Sec17p/Sec18p and HOPS.     

The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking.     

Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion

SNARE functions during membrane docking and fusion are regulated by Sec1/Munc18 (SM) chaperones and Rab/Ypt GTPase effectors. These functions for yeast vacuole fusion are combined in the six-subunit HOPS complex. HOPS facilitates Ypt7p nucleotide exchange, is a Ypt7p effector, and contains an SM protein. We have dissected the associations and requirements for HOPS, Ypt7p, and Sec17/18p during SNARE complex assembly. Vacuole SNARE complexes bind either Sec17p or the HOPS complex, but not both. Sec17p and its co-chaperone Sec18p disassemble SNARE complexes. Ypt7p regulates the reassembly of unpaired SNAREs with each other and with HOPS, forming HOPS.SNARE complexes prior to fusion. After HOPS.SNARE assembly, lipid rearrangements are still required for vacuole content mixing. Thus, Sec17p and HOPS have mutually exclusive interactions with vacuole SNAREs to mediate disruption of SNARE complexes or their assembly for docking and fusion. Sec17p may displace HOPS from SNAREs to permit subsequent rounds of fusion.     

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.     

Fusion with wild-type SNARE domains is controlled by juxtamembrane domains,transmembrane anchors,and Sec17

Membrane fusion requires tethers, SNAREs of R, Qa, Qb, and Qc families, and chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. SNAREs have N-domains, SNARE domains that zipper into 4-helical RQaQbQc coiled coils, a short juxtamembrane (Jx) domain, and (often) a C-terminal transmembrane anchor. We reconstitute fusion with purified components from yeast vacuoles, where the HOPS protein combines tethering and SM functions. The vacuolar Rab, lipids, and R-SNARE activate HOPS to bind Q-SNAREs and catalyze trans-SNARE associations. With SNAREs initially disassembled, as they are on the organelle, we now report that R- and Qa-SNAREs require their physiological juxtamembrane (Jx) regions for fusion. Swap of the Jx domain between the R- and Qa-SNAREs blocks fusion after SNARE association in trans. This block is bypassed by either Sec17, which drives fusion without requiring complete SNARE zippering, or transmembrane-anchored Qb-SNARE in complex with Qa. The abundance of the trans-SNARE complex is not the sole fusion determinant, as it is unaltered by Sec17, Jx swap, or the Qb-transmembrane anchor. The sensitivity of fusion to Jx swap in the absence of a Qb transmembrane anchor is inherent to the SNAREs, because it remains when a synthetic tether replaces HOPS.     

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.     

The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation

The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events. The Vtc complex associates with the R-SNARE Nyv1p and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation. In contrast, subunit Vtc3p is required for the latest step, LMA1 release, but dispensible for all preceding steps, including V(0) trans-complex formation. This suggests that Vtc3p might act close to or at fusion pore opening. We propose that Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0) sectors in order to activate them for V(0) trans-complex formation and/or control fusion pore opening.     

Cdc42p Is Activated during Vacuole Membrane Fusion in a Sterol-dependent Subreaction of Priming

Cdc42p is a Rho GTPase that initiates signaling cascades at spatially defined intracellular sites for many cellular functions. We have previously shown that Cdc42p is localized to the yeast vacuole where it initiates actin polymerization during membrane fusion. Here we examine the activation cycle of Cdc42p during vacuole membrane fusion. Expression of either GTP- or GDP-locked Cdc42p mutants caused several morphological defects including enlarged cells and fragmented vacuoles. Stimulation of multiple rounds of fusion enhanced vacuole fragmentation, suggesting that cycles of Cdc42p activation, involving rounds of GTP binding and hydrolysis, are required to propagate Cdc42p signaling. We developed an assay to directly examine Cdc42p activation based on affinity to a probe derived from the p21-activated kinase effector, Ste20p. Cdc42p was rapidly activated during vacuole membrane fusion, which kinetically coincided with priming subreaction. During priming, Sec18p ATPase activity dissociates SNARE complexes and releases Sec17p, however, priming inhibitors such as Sec17p and Sec18p ligands did not block Cdc42p activation. Therefore, Cdc42p activation seems to be a parallel subreaction of priming, distinct from Sec18p activity. Specific mutants in the ergosterol synthesis pathway block both Sec17p release and Cdc42p activation. Taken together, our results define a novel sterol-dependent subreaction of vacuole priming that activates cycles of Cdc42p activity to facilitate membrane fusion.     

Yeast vacuolar HOPS,regulated by its kinase,exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide–sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.     

Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis

COPII coat proteins are required for direct capture of cargo and SNARE proteins into transport vesicles from the endoplasmic reticulum (ER). Cargo and SNARE capture occurs during the formation of a 'prebudding complex' comprising a cargo, Sar1p-GTP and the COPII subunits Sec23/24p. The assembly and disassembly cycle of the prebudding complex on ER membranes is coupled to the Sar1p GTPase cycle. Using FRET to monitor a single round of Sec23/24p binding and dissociation from SNAREs in reconstituted liposomes, we show that Sec23/24p dissociates from v-SNARE and complexed t-SNARE with kinetics slower than Sar1p-GTP hydrolysis. Once Sec23/24p becomes associated with v-SNARE or complexed t-SNARE, the complex remains assembled during multiple rounds of Sar1p-GTP hydrolysis mediated by the GDP-GTP exchange factor Sec12p. These data suggest a model for the maintenance of kinetically stable prebudding complexes during the Sar1p GTPase cycle that regulates cargo sorting into transport vesicles.     

The docking of primed vacuoles can be reversibly arrested by excess Sec17p (alpha-SNAP)

Homotypic vacuole fusion occurs in ordered stages of priming, docking, and fusion. Priming, which prepares vacuoles for productive association, requires Sec17p (the yeast homolog of alpha-SNAP), Sec18p (the yeast NSF, an ATP-driven chaperone), and ATP. Sec17p is initially an integral part of the cis-SNARE complex together with vacuolar SNARE proteins and Sec18p (NSF). Previous studies have shown that Sec17p is rapidly released from the vacuole membrane during priming as the cis-SNARE complex is disassembled, but the order and causal relationship of these subreactions has not been known. We now report that the addition of excess recombinant his(6)-Sec17p to primed vacuoles can block subsequent docking. This inhibition is reversible by Sec18p, but the reaction cannot proceed to the tethering and trans-SNARE pairing steps of docking while the Sec17p block is in place. Once docking has occurred, excess Sec17p does not inhibit membrane fusion per se. Incubation of cells with thermosensitive Sec17-1p at nonpermissive temperature causes SNARE complex disassembly. These data suggest that Sec17p can stabilize vacuolar cis-SNARE complexes and that the release of Sec17p by Sec18p and ATP allows disassembly of this complex and activates its components for docking.