The Transmembrane Domain of Vam3 Affects the Composition ofcis- and trans-SNARE Complexes to Promote Homotypic Vacuole Fusion

Abstract

It is presently not clear how the function of SNARE proteins is affected by their transmembrane domains.Here, we analyzed the role of the transmembrane domain of the vacuolar SNARE Vam3 by replacing it by a lipid anchor. Vacuoles with mutant Vam3 fuse poorly and have increased amounts of cis-SNARE complexes, indicating that they are more stable. As a consequence efficient cis-SNARE complex disassembly that occurs at priming as a prerequisite of fusion requires addition of exogenous Sec18. trans-SNARE complexes in this mutant accumulate up to 4-fold over wild type, suggesting that the transmembrane domain of Vam3 is required to transit through this step. Finally, palmitoylation of Vac8, a reaction that also occurs early during priming is reduced by almost one-half. Since palmitoylated Vac8 is required beyond trans-SNARE complex formation, this may partially explain the fusion deficiency.Membrane fusion along the secretory pathway requires the specific interaction of SNAREs 1 that are localized to vesicles and target organelles (1). Fusion reactions are controlled by Sec18/NSF and ␣-SNAP/yeast Sec17 that disassemble preexisting SNARE complexes or activate t-SNAREs (2, 3). Upon docking of a vesicle with its target membrane, SNAREs from opposite membranes form trans-SNARE complexes, which are a prerequisite for complete fusion (4). In vitro studies with recombinant SNAREs, reconstituted into liposomes, indicated that SNAREs can drive bilayer mixing by pairing in a cognate fashion (5, 6).t-SNAREs, also termed syntaxins, consist of three domains: the regulatory N terminus, the coiled-coil domain required for SNARE complex formation (7-11), and the transmembrane domain (TMD), by which most SNAREs are anchored to membranes (12). Transmembrane domains of SNAREs are important for SNARE function. Alterations in synaptobrevin or syntaxin TMDs in Caenorhabditis elegans cause strong neurotransmission defects (13). C. elegans and vertebrates contain syntaxin isoforms that differ only by their TMDs (14 -16). Furthermore, TMDs of syntaxin 1A and synaptobrevin II drive homo-as well as heterodimerization (17)(18)(19). Moreover, the interaction of syntaxin 1A with synaptobrevin, but not SNAP- (24), suggesting that they constitute autonomous fusogenic domains. However, the evidence suggesting that SNAREs themselves may act as catalysts relies mostly on minimal systems. It does not exclude the possibility that SNAREs regulate other factors that could act as fusogens on cellular membranes or enhance SNARE-mediated fusion (25)(26)(27). 25, depends on the TMD of the t-SNARE (20). In addition, in vitro fusion of liposomes requires anchoring of SNAREs via transmembrane domains (21) and tight coupling between the coiled-coil and the TMD (22). Replacing the TMDs of exocytic SNAREs in yeast arrested secretion (23). Interestingly, TMD peptides alone are sufficient to drive fusion of liposomesYeast vacuole fusion occurs in a cascade of priming, docking, and fusion (28). Five SNAREs, the t-SNARE-like proteins Vam3, Vam7, and Vti1, and the v-SNA...

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

“…Although our findings are consistent with numerous data attributing a key role for the TMD of SNARE proteins in promoting membrane fusion and fusion pore opening (23)(24)(25)(26)(27)(28)(29)51), they also extend our understanding about how SM proteins can assist lipid-anchored SNAREs in mediating complete membrane fusion. In these lines, these data agree with a report showing that addition of the yeast HOPS complex, which includes the SM protein VPS33, restores complete fusion in a liposome-based fusion reaction containing reconstituted lipid-anchored v-SNARE, Nyv1p (27).…”

“…When the TMD of the HA protein is replaced with glycosylphosphatidylinositol (GPI), a lipid anchor that spans only the outer leaflet of the membrane, membrane fusion ends in a hemifusion state where the outer monolayers of the membranes are fused, whereas the inner monolayers and the aqueous contents remain segregated (20)(21)(22). Similarly, replacement of the TMD of SNARE proteins by a lipid or protein anchor that spans a single leaflet of the bilayer inhibited complete fusion in vitro (23)(24)(25)(26)(27) and in vivo (28). Crystallographic analysis of a neuronal SNARE complex containing the TMDs revealed that SNARE motifs and the TMDs form continuous interacting α-helices (29), potentially explaining the role of the TMD in full fusion.…”

A lipid-anchored SNARE supports membrane fusion

“…Because equal amounts of Vam3p were immunoprecipitated and there had been equal inputs of Nyv1p and Nyv1p-CCIIM, we conclude that the lipid-anchored Nyv1p can effectively form trans-SNARE complexes. These trans-SNARE complexes alone cannot support lipid mixing, in accord with other reports that lipid-anchored SNAREs can form nonfunctional or potentially inhibitory SNARE complexes (9,22,27).…”

Yeast vacuole fusion: A model system for eukaryotic endomembrane dynamics

“…41 Indeed, fusion-dependent release of Ca 2+ also occurs if vacuoles lacking Nyv1 are incubated with vam3D vacuoles, which had previously been used for trans-SNARE complex detection. 41,53,71,100,103,104 Fusion requires transmembrane-anchored Vam3, 105 and fusion efficiency has been connected to the structure of the Vam3 transmembrane domain. 106 Furthermore, if the coupling between transmembrane domain and SNARE domain was impaired by the insertion of 12 amino acids, fusion was reduced to background levels.…”