SNAP receptor (SNARE) proteins function in intracellular trafficking by forming complexes that bridge vesicle and target membranes prior to fusion. Biochemical studies indicate that the entry of certain SNARE proteins into complexes is inhibited by intramolecular interactions that generate a closed conformation. For example, an essential N-terminal regulatory domain of the yeast plasma membrane SNARE Sso1p sequesters the C-terminal SNARE motif and prevents it from binding to its assembly partners Sec9p and Sncp. Here, we introduce mutations into Sso1p that cause it to remain constitutively open. These open mutants can functionally substitute for wild-type Sso1p protein in vivo, demonstrating that inhibition of SNARE assembly is not the essential function of the N-terminal regulatory domain. Furthermore, the open mutants suppress sec9--4, a mutation that causes a severe defect in SNARE assembly. Elevated levels of SNARE complexes are observed in cells expressing the open mutants. In the presence of sufficient Sec9p, these complexes accumulate to levels that cause severe growth defects. Similarly, overexpression of the open mutants in yeast carrying mutations in the SNARE disassembly machinery impairs growth. Our findings indicate that elevated levels of SNARE complexes can be toxic and that these levels are normally controlled by the SNARE disassembly machinery, by the limited availability of Sec9p, and by the closed conformation of Sso1p.
"In this closed conformation, the syntaxin is unable to form SNARE complexes, owing to inaccessibility of the SNARE motif. The SNARE motif is released from this inhibition in the open conformation, allowing SNARE-complex assembly to proceed (Dulubova et al., 1999; Misura et al., 2000; Munson and Hughson, 2002). "
[Show abstract][Hide abstract] ABSTRACT: Membrane fusion in all eukaryotic cells is regulated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. The molecular mechanisms that control this process are conserved through evolution and require several protein families, including Sec1p/Munc18 (SM) proteins. Here, we demonstrate that the mammalian SNARE protein syntaxin 16 (Sx16, also known as Syn16) is a functional homologue of the yeast SNARE Tlg2p, in that its expression fully complements the mutant phenotypes of tlg2Delta mutant yeast. We have used this functional homology to demonstrate that, as observed for Tlg2p, the function of Sx16 is regulated by the SM protein Vps45p. Furthermore, in vitro SNARE-complex assembly studies demonstrate that the N-terminal domain of Tlg2p is inhibitory to the formation of SNARE complexes, and that this inhibition can be lifted by the addition of purified Vps45p. By combining these cell-biological and biochemical analyses, we propose an evolutionarily conserved regulatory mechanism for Vps45p function. Our data support a model in which the SM protein is required to facilitate a switch of Tlg2p and Sx16 from a closed to an open conformation, thus allowing SNARE-complex assembly and membrane fusion to proceed.
"Our interpretation of these results is that mutations in SEC9 affect a constitutive function, but that the temperature-sensitive phenotype is a manifestation of greater cellular requirement for this absent function under the increased physiological demands of higher temperature. This is consistent with previous observations that the level of SNARE complexes can be controlled by the availability of Sec9p, suggesting that Sec9p is limiting for SNARE complex formation . Suppressing conditions decrease physiological demands by slowing the growth rate and thereby bring the cells back to normal homeostasis at elevated temperatures. "
[Show abstract][Hide abstract] ABSTRACT: Growth and division of Saccharomyces cerevisiae is dependent on the action of SNARE proteins that are required for membrane fusion. SNAREs are regulated, through a poorly understood mechanism, to ensure membrane fusion at the correct time and place within a cell. Although fusion of secretory vesicles with the plasma membrane is important for yeast cell growth, the relationship between exocytic SNAREs and cell physiology has not been established.
Using genetic analysis, we identified several influences on the function of exocytic SNAREs. Genetic disruption of the V-ATPase, but not vacuolar proteolysis, can suppress two different temperature-sensitive mutations in SEC9. Suppression is unlikely due to increased SNARE complex formation because increasing SNARE complex formation, through overexpression of SRO7, does not result in suppression. We also observed suppression of sec9 mutations by growth on alkaline media or on a non-fermentable carbon source, conditions associated with a reduced growth rate of wild-type cells and decreased SNARE complex formation.
Three main conclusions arise from our results. First, there is a genetic interaction between SEC9 and the V-ATPase, although it is unlikely that this interaction has functional significance with respect to membrane fusion or SNAREs. Second, Sro7p acts to promote SNARE complex formation. Finally, Sec9p function and SNARE complex formation are tightly coupled to the physiological state of the cell.
PLoS ONE 02/2009; 4(5):e5449. DOI:10.1371/journal.pone.0005449 · 3.23 Impact Factor
"Vol. 20, February 1, 2009 979 would rescue this defect, because it readily forms SNARE complexes. However, similarly to the synthetic defects observed in combination with other exocyst ts mutants, this mutant failed to suppress the sec6-49 and -54 ts phenotypes (Figure 5E), likely because of a detrimental overaccumulation of SNARE complexes (Munson and Hughson, 2002). In contrast, the sec6-4 allele, in which the exocyst complex is completely disassembled (TerBush and Novick, 1995), has a more severe block in SNARE complex assembly (Grote et al., 2000) that cannot be suppressed by overexpression of SSO1 and SEC9 (Aalto et al., 1993). "
[Show abstract][Hide abstract] ABSTRACT: The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two "patches" of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.
Molecular biology of the cell 01/2009; 20(3):973-82. DOI:10.1091/mbc.E08-09-0968 · 4.47 Impact Factor
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