Abstract
In Saccharomyces cerevisiae, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity. Ras proteins also have PKA-independent functions in mitosis and actin repolarization. We have found that mutations in MOB2 or CBK1 confer a slow-growth phenotype in a ras2Delta background. The slow-growth phenotype of mob2Delta ras2Delta cells results from a G1 delay that is accompanied by an increase in size, suggesting a G1/S role for Ras not previously described. In addition, mob2Delta strains have imprecise bud site selection, a defect exacerbated by deletion of RAS2. Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes. The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1. From these observations, we conclude that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression.
Highlights
Ras proteins are highly conserved, small monomeric GTPases that function as molecular switches in signal transduction pathways to regulate cell growth and differentiation in response to various environmental cues
Deletion of the Ras-related GTPase RSR1 in a ras1⌬ ras2⌬ strain results in a late mitotic arrest that cannot be suppressed by TPK1 overexpression but can be suppressed by overexpression of any of a number of genes involved in mitotic exit, including DBF2, CDC5, CDC15, and SPO12 [38]
The MOB2/CBK pathway is required for efficient cell cycle progression in a ras2⌬ strain
Summary
Exponentially growing cells (2 ml) were formaldehyde fixed (3.7% final concentration) and stained with calcofluor white (Fluorescent Brightener 28; Sigma; final concentration, 0.17 mg/ml) as described previously [6]. To examine actin polarization in mitotic cells, exponentially growing cells were fixed and stained with rhodamine phalloidin (2.2 M; Molecular Probes) followed by staining with calcofluor white as described previously [6]. Cells were grown in YEPD or SC-LEU and synchronized with ␣-factor (5 g/ml) as described previously [3]. Cells were fixed as described previously [6], treated with RNase A (0.25 mg/ml in sodium citrate buffer [pH 7.2] overnight at 37°C) and proteinase K (2 mg/ml; 2 h at 50°C), and stained with Sytox green (1 M in sodium citrate [pH 7.2]; Molecular Probes). Signal intensity was quantified using a STORM 860 imaging system (Molecular Dynamics)
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