mob2Δ confers a synthetic slow-growth phenotype to a ras2Δ strain. (A) Strain Y3011 (ras2 mob2 [CEN-RAS2-URA3]) harboring, from top to bottom, [CEN-LEU2], [CEN-LEU2-RAS2], [CEN-HIS3], or [CEN-HIS3-MOB2] were grown in SC liquid medium, spotted onto SC and SC plus FOA plates in fivefold serial dilutions, and incubated at 30°C. (B) Tetrad analysis of Y3012 (mob2Δ) × Y3013 (ras2Δ). A representative tetratype tetrad is labeled and shown at the left of the tetrads. At least 10 tetrads were analyzed with consistent results (mob2Δ ras2Δ spore clones are circled). (C) cbk1Δ, hym1Δ, pag1Δ, and kic1Δ confer a synthetic slow-growth phenotype with ras2Δ. In all subpanels, representative tetratype tetrads are labeled and shown. For each strain, at least 10 tetrads were analyzed with results fully consistent with those shown. (Left to right) Tetrad analysis of Y3013 (ras2Δ) × Y3016 (cbk1Δ), Y3013 (ras2Δ) × Y3027 (hym1Δ), Y3090 (ras2Δ) × Y3088 (pag1Δ), and Y2813 (ras2Δ) × Y3089 (kic1Δ).

Overexpression of TPK1 suppresses the mob2Δ ras2Δ and cbk1Δ ras2Δ slow-growth phenotypes. (A) Y3018 (ras2Δ mob2Δ [CEN-URA3-RAS2]) and Y3019 (ras2Δ cbk1Δ [CEN-URA3-RAS2]) were transformed with YEp13 (LEU2 2μm) and B1373 (LEU2 TPK1 2μm) and analyzed for growth on SC-Leu and SC-Leu plus FOA as described in the legend to Fig. 1A. (B) Representative tetratype tetrad from tetrad analysis of a diploid resulting from crossing Y3013 and Y3017. More than 10 tetrads were analyzed with consistent results.

The slow growth of a mob2Δ ras2Δ strain is not due to loss of Ace2 localization or activation. (A) Northern blot of CTS1 and ACT1 RNA from strains Y3020 (wild type [WT]), Y3021 (ras2/ras2), Y3022 (mob2/mob2), and Y3023 (ras2/ras2 mob2/mob2) transformed with plasmids carrying the indicated markers (YEp13, B1373, B2461, or B2462). Normalized values for the levels of CTS1 mRNA are indicated below the Northern blot. (B) Y3018 (ras2Δ mob2Δ [CEN-URA3-RAS2]) containing a plasmid carrying the indicated marker was spotted onto SC and SC plus FOA as for Fig. 1A.

Filamentous actin is polarized in a mob2Δ ras2Δ strain. Calcofluor white (upper panels) and rhodamine phalloidin (lower panels) staining of strains Y3020 (wild type [WT]), Y3021 (ras2Δ/ras2Δ), Y3022 (mob2Δ/mob2Δ), and Y3023 (ras2Δ/ras2Δ mob2Δ/mob2Δ) is shown. Bars = 5 μm.

Morphological changes and formation of mating projections in response to pheromone. Calcofluor white (first and third columns) and rhodamine phalloidin (second and fourth column) staining of strains Y3014 (wild type [WT]), Y3025 (mob2Δ), and Y3015 (mob2Δ ras2Δ) containing Yep13 (vector) or B1373 (TPK1 2μm) and treated with α-factor as described in Materials and Methods is shown.

Slow-growth phenotype of mob2Δ ras2Δ strains is caused by a growth delay, not loss in viability. (A) Differential interference contrast (left panels) and FUN 1 (right panels) staining of wild-type strain Y3014 (upper panels) and mob2Δ ras2Δ strain Y3015 (lower panels). (B) Percent cells (n ≥ 200) exhibiting positive staining with FUN 1 as measured by the appearance of cylindrical intracellular vacuolar structures (CIVS).

The slow growth of a mob2Δ ras2Δ strain is due to a G₁ delay. (A) Strains Y3084 (wild type [WT]), Y3085 (mob2Δ), Y3086 (ras2Δ), and Y3087 (mob2Δ ras2Δ) were synchronized in G₁ as described in Materials and Methods. Cells were collected at the indicated times (minutes), stained with Sytox green, and analyzed by fluorescence-activated cell sorting (FACS) as described in Materials and Methods. (B) Strain Y3087 (mob2Δ ras2Δ) was transformed with YEp13 (vector) or B1373 (TPK1 2μm) and synchronized in G₁ as described in Materials and Methods. Cells were collected at the indicated times, stained with Sytox green, and analyzed by FACS as described in Materials and Methods. (C) Percentages of budded cells as a function of time following release from α-factor block. ▪, wild type (Y3084); •, mob2Δ (Y3085); ▴, mob2Δ ras2Δ (Y3087). (D) Forward scatter of asynchronous Y3084, Y3085, Y3086, and Y3087 fixed cells. (E) Cells of the indicated strains were synchronized with α-factor and then plated on YEPD and examined by time-lapse microscopy over two generations. Shown is the cumulative percent mother cells with the indicated time interval between the emergence of the first and second buds. (F) Shown is the cumulative percent daughter cells from the experiment for which results are shown in panel E with the indicated interval between birth and emergence of the first bud.

Impairment of the morphogenesis checkpoint exacerbates the slow-growth phenotype of a mob2Δ ras2Δ strain. (Left) Tetrad analysis of Y3154 (swe1Δ ras2Δ) × Y3157 (mob2Δ ras2Δ). Genotypes of spore clones are shown below the panel (MOB2, circle; mob2, diamond; SWE1, filled symbol; swe1, open symbol). (Right) Tetrad analysis of Y3157 (mob2Δ ras2Δ) × Y3155 (cdc28 ^Y19F ras2Δ). Individual segregants were analyzed by PCR amplification and KpnI digestion of the CDC28 locus to determine the presence of the cdc28^Y19F mutation. Genotypes of spore clones are shown below the panel (MOB2, circle; mob2 diamond; CDC28, filled symbol; cdc28^Y19F, open symbol).

Model for the roles of Ras and Mob2 in S. cerevisiae. Ras proteins have PKA-independent functions in mitotic exit and actin repolarization and PKA-dependent functions affecting metabolism, transcription, and cell growth. Cbk1, together with Mob2, regulates the Ace2 transcription factor, effecting daughter cell-specific gene expression. Cbk1 and Mob2 have additional Ace2-independent functions in regulating apical growth and mating projection formation. Hym1 interacts with Kic1 and Sog2, and this complex along with Pag1 interacts with Cbk1 to promote its localization and function. Data presented in this report demonstrate that the Ras/PKA and Cbk1/Mob2 pathways play parallel roles in cell growth and bud site selection, likely through regulation of Rho1 (see Discussion for details). Cbk1 and the Ras/PKA pathway independently repress cellular trehalose levels through a different mechanism.