Analysis of SEC9 Suppression Reveals a Relationship of SNARE Function to Cell Physiology

BACKGROUND: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. METHODOLOGY/PRINCIPAL FINDINGS: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. CONCLUSIONS/SIGNIFICANCE: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

Myosin Vs organize actin cables in fission yeast.

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces