Abstract
BackgroundGrowth 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.Methodology/Principal FindingsUsing 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.Conclusions/SignificanceThree 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.
Highlights
Cell growth and division requires the addition of membrane and protein to the surface of the growing cell through the fusion of secretory vesicles with the plasma membrane [1,2]
We reasoned that mutations that restore growth to sec9-4 cells at the non-permissive temperature might do so by increasing SNARE complex formation
The growth characteristics of sec9-4 strains harboring SRO7 on a high-copy number plasmid were indistinguishable from empty vector controls at permissive and nonpermissive temperatures (Figure 4c). These results demonstrate that increased SNARE complex formation does not result in suppression of sec9-4 and suggest that the suppression of sec9 mutations, by loss of VMA function or increased pH of the media, is unlikely the result of increased SNARE assembly
Summary
Cell growth and division requires the addition of membrane and protein to the surface of the growing cell through the fusion of secretory vesicles with the plasma membrane [1,2]. The sec mutation encodes a Gly to Asp amino acid substitution in the N-terminal helical domain of Sec9p that reduces the ability of Sec9-4p to complex with Sso1/2p and Snc1/2p [8]. Another temperature-sensitive allele (sec9-7) results in a lower cut-off temperature, but does not affect SNARE complex formation in vitro [9] suggesting multiple functions for Sec9p. 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
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