The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes

The fusion of yeast vacuolar membranes depends on the disassembly of cis–soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles.     

Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p

Coupling of Rab GTPase activation and SNARE complex assembly during membrane fusion is poorly understood. The homotypic fusion and vacuole protein sorting (HOPS) complex links these two processes: it is an effector for the vacuolar Rab GTPase Ypt7p and is required for vacuolar SNARE complex assembly. We now report that pure, active HOPS complex binds phosphoinositides and the PX domain of the vacuolar SNARE protein Vam7p. These binding interactions support HOPS complex association with the vacuole and explain its enrichment at the same microdomains on docked vacuoles as phosphoinositides, Ypt7p, Vam7p, and the other SNARE proteins. Concentration of the HOPS complex at these microdomains may be a key factor for coupling Rab GTPase activation to SNARE complex assembly.     

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.     

Distinct targeting and fusion functions of the PX and SNARE domains of yeast vacuolar Vam7p

Regulated membrane fusion requires organelle tethering, enrichment of selected proteins and lipids at the fusion site, bilayer distortion, and lipid rearrangement. Yeast vacuole homotypic fusion requires regulatory lipids (ergosterol, diacylglycerol, and phosphoinositides), the Rab family GTPase Ypt7p, the multisubunit Ypt7p-effector complex HOPS (homotypic fusion and vacuole protein sorting), and four SNAREs. One SNARE, Vam7p, has an N-terminal PX domain which binds to phosphatidylinositol 3-phosphate (PI(3)P) and to HOPS and a C-terminal SNARE domain but no apolar membrane anchor. We have exploited an in vitro reaction of vacuole fusion to analyze the functions of each domain, removing the PX domain or mutating it to abolish its PI(3)P affinity. Lowering the PI(3)P affinity of the PX domain, or even deleting the PX domain, affects the fusion K(m) for Vam7p but not the maximal fusion rate. Fusion driven by the SNARE domain alone is strikingly enhanced by the PLC inhibitor U73122 through enhanced binding of Vam7p SNARE domain to vacuoles, and the further addition of Plc1p blocks this U73122 effect. The PX domain, through its affinities for phosphoinositides and HOPS, is thus exclusively required for enhancing the targeting of Vam7p rather than for execution of the Vam7p functions in HOPS.SNARE complex assembly and fusion.     

The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion

Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the H_abc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 H_abc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the H_abc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the H_abc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the H_abc domain. In agreement, membranes carrying Vam3ΔH_abc fuse poorly unless an excess of HOPS is provided. These data suggest that the H_abc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.     

HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites

Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.     

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.     

Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H_abc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.     

The N-terminal Domain of the t-SNARE Vam3p Coordinates Priming and Docking in Yeast Vacuole Fusion

Homotypic fusion of yeast vacuoles requires a regulated sequence of events. During priming, Sec18p disassembles cis-SNARE complexes. The HOPS complex, which is initially associated with the cis-SNARE complex, then mediates tethering. Finally, SNAREs assemble into trans-complexes before the membranes fuse. The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes. We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion. The truncated protein (Vam3 Delta N) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain. However, in vitro vacuole fusion is strongly reduced due to the following reasons: Assembly, as well as disassembly of the cis-SNARE complex is more efficient on Vam3 Delta N vacuoles; however, the HOPS complex is not associated well with the Vam3 Delta N cis-complex. Thus, primed SNAREs from Vam3 Delta N vacuoles cannot participate efficiently in the reaction because trans-SNARE pairing is substantially reduced. We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion.     

The Major Role of the Rab Ypt7p in Vacuole Fusion Is Supporting HOPS Membrane Association

Yeast vacuole fusion requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), the Rab GTPase Ypt7p, vacuolar lipids, Sec17p and Sec18p, and the homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a multisubunit protein with direct affinities for SNAREs, vacuolar lipids, and the GTP-bound form of Ypt7p; each of these affinities contributes to HOPS association with the organelle. Using all-purified components, we have reconstituted fusion, but the Rab Ypt7p was not required. We now report that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1–46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, although Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.Rab proteins are small GTP-binding proteins involved in multiple steps of membrane traffic, including protein sorting, vesicle transport, and SNARE³-dependent membrane fusion (1). Rabs in their GTP-bound state bind proteins that are essential for mediating Rab function, which are therefore termed “effectors.” These effectors are diverse and perform various biochemical functions. For membrane fusion, Rabs and their effectors support tethering, the initial membrane contact that is needed for the subsequent assembly of trans-SNARE complexes between membranes (1, 2). A central question in organelle trafficking, which we now address, is whether Rabs are only required for binding their effectors to the membrane or whether they also activate the bound effector or provide some additional essential function for membrane fusion.We study membrane fusion using isolated yeast vacuoles (3). Yeast vacuole fusion requires the Rab GTPase Ypt7p, the heterohexameric HOPS complex, four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, and chemically minor yet functionally essential lipids, termed “regulatory” lipids. The HOPS complex is an effector of Ypt7p (4) and belongs to a group of functionally conserved large multisubunit tethering complexes, many of which are Rab effectors (5). The Vps39p subunit of HOPS is a nucleotide exchange factor for Ypt7p (6). HOPS is also a SNARE chaperone; its Vps33p subunit is a Sec1p/Munc18-1 family (SM) protein, HOPS binds multiple vacuolar SNAREs (7–9), and it proofreads SNARE complex structure (10). HOPS also binds to specific phosphoinositides (8), and these are among the regulatory lipids that are important for fusion (11–13).We have recently reconstituted membrane fusion using proteoliposomes of pure vacuolar proteins and lipids (13). HOPS and the regulatory lipids are crucial for rapid fusion of proteoliposome pairs bearing the three Q-SNAREs on one proteoliposome and the R-SNARE on the other and are absolutely required when all four SNAREs are present on each proteoliposome and Sec17p and Sec18p are present. Ypt7p is not required, showing that HOPS can stimulate SNARE-dependent fusion in vitro even in the absence of its Rab, although Ypt7p stimulates the fusion of these proteoliposomes.4Yeast vacuole fusion can be negatively regulated either by GTPase-activating proteins (GAPs) (14, 15) that promote GTP hydrolysis by Ypt7p or by the kinase Yck3p, which phosphorylates the Vps41p subunit of HOPS (16) and the vacuolar SNARE Vam3p (15). Yck3p is a palmitoylated (17), vacuole-localized kinase of the casein kinase I family (18). The complete fragmentation of vacuoles in vivo, indicating a block of fusion, requires both Ypt7p inactivation by a RabGAP and the presence of Yck3p (15). Yck3p is necessary for efficient vacuole inheritance (16) and normal vacuole morphology (19), suggesting that its function is part of the normal mechanism of vacuole segregation during the cell cycle. Although Yck3p clearly regulates vacuole fusion through phosphorylation of HOPS, it remains unclear which activities of HOPS are inhibited by Yck3p phosphorylation and whether Yck3p must also phosphorylate other vacuole fusion proteins such as Vam3p to block fusion.We now show that phosphorylation of the Vps41p subunit of HOPS by purified Yck3p reduces HOPS binding to membrane lipids, thereby making HOPS association with the membrane and the ensuing fusion of reconstituted proteoliposomes dependent on active Ypt7p. These data with proteoliposomes are supported by assays with purified vacuoles; the RabGAP Gyp1–46p only inhibits the in vitro fusion of yck3Δ vacuoles when purified Yck3p is added. As for Ypt7p and HOPS, the major function of other Rabs may also be to act as membrane receptors for their effectors.     

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.     

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.     

HOPS proofreads the trans-SNARE complex for yeast vacuole fusion

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.     

Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion

The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle-Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)-this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.     

Molecular mechanism of membrane docking by the Vam7p PX domain

The Vam7p t-SNARE is an essential component of the vacuole fusion machinery that mediates membrane trafficking and protein sorting in yeast. Vam7p is recruited to vacuoles by its N-terminal PX domain that specifically recognizes PtdIns(3)P in the bilayers, however the precise mechanism of membrane anchoring remains unclear. Here we describe a molecular basis for membrane targeting and penetration by the Vam7p PX domain based on structural and quantitative analysis of its interactions with lipids and micelles. Our results derived from in vitro binding measurements using NMR, monolayer surface tension experiments and mutagenesis reveal a multivalent membrane docking mechanism involving specific PtdIns(3)P recognition that is facilitated by electrostatic interactions and accompanying hydrophobic insertion. Both the hydrophobic and electrostatic components enhance the Vam7p PX domain association with PtdIns(3)P-containing membranes. The inserting Val(70), Leu(71), and Trp(75) residues located next to the PtdIns(3)P binding pocket are surrounded by a basic patch, which is involved in nonspecific electrostatic contacts with acidic lipids, such as PtdSer. Substitution of the insertion residues significantly reduces the binding and penetrating power of the Vam7p PX domain and leads to cytoplasmic redistribution of the EGFP-tagged protein. The affinities of the PX domain for PtdIns(3)P and other lipids reveal a remarkable synergy within the multivalent complex that stably anchors Vam7p at the vacuolar membrane.     

Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein-protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble "vertex" ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)-VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the "regulatory lipids" ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.     

LegC3, an Effector Protein from Legionella pneumophila,Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.     

The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter‐membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low‐curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p‐bearing low‐curvature liposomes even when the high‐curvature liposomes are protein‐free. Phosphorylation of the curvature‐sensing amphipathic lipid‐packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high‐curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high‐curvature liposomes and Ypt7p‐bearing low‐curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein–membrane interaction. Such high‐curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole‐vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high‐curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.      

HOPS Interacts with Apl5 at the Vacuole Membrane and Is Required for Consumption of AP-3 Transport Vesicles

Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 δ subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or 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.