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.
Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.biochemical reconstitution ͉ Rab effector
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 GTPaseactivating 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 SNARE 3 -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 SNAR...
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