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

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Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE-proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A lipid-anchored SNARE supports membrane fusion

Zick²,

Wickner³

et al. 2011

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“…Although it is possible that certain components, such as tethering factors, prevent cis interactions, it would be important to test for the formation of cis-SNARE complexes with supported bilayers, as done here for ATL. Futile tethering cycles also may be relevant to SNAREmediated fusion, given that trans-SNARE complexes can be disassembled before they cause fusion (29). Finally, as in the case of ATL, fusion is facilitated when multiple SNARE complexes assemble simultaneously in trans (30).…”

Section: Discussionmentioning

confidence: 99%

Cis and trans interactions between atlastin molecules during membrane fusion

Liu

Bian

Romano

et al. 2015

115

Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions. T he endoplasmic reticulum (ER) consists of tubules and sheets connected into a characteristic network by membrane fusion (1-3). In contrast to heterotypic fusion between viral and cellular membranes or between intracellular transport vesicles and target membranes, the fusing ER membranes are identical (i.e., homotypic fusion). The mechanism of heterotypic fusion has been studied extensively, leading to the concept that a conformational change of a viral fusion protein or the assembly of SNARE proteins pulls two opposing membranes together so that they can fuse (4-7). Recently, some insight has been obtained into homotypic ER fusion as well. This process is mediated by the atlastins (ATLs) in metazoans and by Sey1p/ROOT HAIR DEFECTIVE3 (RHD3)-related proteins in yeast and plants (8,9).The ATLs and Sey1p/RHD3 are membrane-bound GTPases that belong to the dynamin family (reviewed in refs. 10, 11). They contain cytosolic N-terminal GTPase (G) and helical bundle domains, followed by two closely spaced transmembrane (TM) segments and a cytosolic C-terminal tail (12)(13)(14). A role for the GTPases in ER fusion is suggested by the observation that their depletion or mutational inactivation leads to long, nonbranched tubules or fragmented ER (8, 9). Nonbranched ER tubules are also observed on expression of dominant-negative ATL mutants (8,12). In addition, the fusion of ER vesicles in Xenopus laevis egg extracts is prevented by ATL antibodies or a cytosolic fragment of ATL (8,15), and the fusion of the ER in mating yeast cells is delayed on SEY1 deletion (16). Most convincingly, proteoliposomes containing purified ATL, Sey1p, or RHD3 undergo GTP-dependent fusion in vitro (9,13,16,17). Defects in ATL-mediated ER fusion can cause hereditary spastic paraplegia, a neurodegenerativ...

“…(A) NBD dequenching, expressed as its ratio over the fluorescent signal at t = 0. At the end of the 15-min incubation, samples were transferred to ice, solubilized in RIPA buffer, and subjected to immunoprecipitation using immobilized antiVam3p antibody (21). Western blotting of the immunoprecipitated Vam3p and the coprecipitated Nyv1p are shown in B.…”

Section: Methodsmentioning

confidence: 99%

“…Vacuole fusion requires Nyv1p, the R-SNARE (in reference to the arginyl residue at the center of its SNARE domain), and three Q-SNAREs (with central glutamyl residues), Vam3p, Vti1p, and Vam7p, members of the conserved Qa, Qb, and Qc families (15,16), respectively. cis-SNARE complexes, with each SNARE anchored to the same membrane, are disassembled by the chaperones Sec18p and Sec17p, permitting the individual SNAREs to participate in the assembly of trans-SNARE complexes (17)(18)(19)(20)(21). The roles of the SNARE N-domains, whether for localization to the fusion ring microdomain or for SNARE complex assembly, are largely unknown.…”

mentioning

confidence: 99%

N-terminal domain of vacuolar SNARE Vam7p promotes trans -SNARE complex assembly

Wickner

2012

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SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R-and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R-and Q-SNAREs. This function of the Vam7p Ndomain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain.phosphoinositide | Phox homology domain M embranes fuse by conserved mechanisms during endocytic and exocytic vesicular trafficking (1). The initial step of association between membranes, termed tethering, is mediated by a Rab-family GTPase and its associated tethering proteins (2). Selected proteins and lipids then become enriched in a fusioncompetent microdomain. SNARE proteins anchored to apposed membranes form four-helix bundles, the trans-SNARE complex, in conjunction with SNARE-binding factors such as Sec1-Munc18 proteins and, at neuronal synapses, Munc13, complexin, and synaptotagmin (3, 4). Although SNARE proteins are central to the ensuing fusion, little is known of the roles of their N-domains.We study fusion mechanisms with yeast vacuoles (lysosomes) (5). Vacuoles undergo constant homotypic fusion and fission in the cell in response to the osmolarity of the growth environment. When fusion is genetically impaired, continuing fission causes a fragmented vacuole morphology, the vam phenotype (6), which allowed identification of the genes for proteins that catalyze vacuole fusion. Their roles were confirmed and extended through studies of the fusion of the isolated organelle (7). Vacuole clustering (tethering) is mediated by the Rab GTPase Ypt7p through its direct binding to the hexameric homotypic fusion and vacuole protein sorting (HOPS) complex (8-11). As vacuole membranes are drawn together, the proteins and lipids needed for fusion become enriched in a ring-shaped microdomain whic...