Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle

Abstract

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was ratelimiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.

The C Terminus of the Vps34p Phosphoinositide 3-Kinase Is Necessary and Sufficient for the Interaction with the Vps15p Protein Kinase

“…The cells were again subjected to a short centrifugation and resuspended in 1 ml of spheroplasting buffer (10 mM KP i , pH 7.4, 2.1 M sorbitol). Zymolyase-20T (Seikagaku) was added to 80 g/ml, and the cells were incubated for 30 min at 30°C (35). The spheroplasts formed were collected by centrifugation for 2 min at 4,000 ϫ g. The spheroplasts were then lysed by the addition of 1 ml of ice-cold TBS buffer (100 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing protease inhibitors.…”

Synaptonemal complex formation and meiotic checkpoint signaling are linked to the lateral element protein Red1

“…Samples from synchronously growing cultures were harvested at the indicated times and fixed in ethanol for subsequent DAPI staining. Symbols are identical to those used in C. (C) For germination assays, spores were isolated and zymolasetreated after 72 h in sporulation media and then plated on YDP plates at 30°C, as described previously (43). For B and C, the Y1083, Y2031, CE571, and CE579 strains were used.…”

Existence of a tightly regulated water channel in Saccharomyces cerevisiae

“…Sporulation was carried out by harvesting the S1278b or W303-1A diploid strains entering the stationary growth phase in YPD, washing the cells in distilled water and placing them in 1% potassium acetate, at 30 8C, under vigorous agitation. After 6 days, the spores were visualized by light microscopy and purified according to the method described by Herman and Rine [5], and used to inoculate a YPD medium where germination and growth occurred.…”