GTPase-activating proteins for Cdc42

Abstract

The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of RGA1, but not RGA2 or BEM3, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of BEM3 resulted in cells with severe morphological defects not observed in rga1delta or rga2delta strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.

FIG. 1.

Protein sequence alignments of Rho-GAPs in yeast. (A) Protein alignment of putative GAP motifs for Rho-type GTPases in the yeast S. cerevisiae. Consensus residues are shaded for residues shared by seven or eight of the proteins and unshaded for residues shared by four to six of the proteins. ∗∗, 61 omitted residues for Sac7. (B) A graphical representation of the known and putative GAPs for Cdc42. The numbers correspond to amino acid positions in the protein sequence. I, identity; S, similarity; LIM, tandem LIM domains; PH, a pleckstrin homology domain; Rho-GAP, consensus GAP motif as shown in panel A.

FIG. 2.

GAP domains of Rga1, Rga2, and Bem3 display biochemical GAP activity for Cdc42, but not for Rsr1. The relative percentage of [γ-³²P]GTP bound to GST-Cdc42^C188S (A), [α-³²P]GTP bound to GST-Cdc42^C188S (B), [γ-³²P]GTP bound to GST-Cdc42^{Q61L, C188S} (C), and [γ-³²P]GTP bound to GST-Rsr1 when incubated with ∼2 μg of purified MBP, MBP-Rga1 GAP, MBP-Rga2 GAP, or MBP-Bem3 GAP (D). The ratio of released counts (³²P_i) to counts still bound to protein is plotted as a function of time. (A) Shown is a representative graph from multiple data sets.

FIG. 3.

Septin localization in normal, peanut, and fingered cells. Septins were visualized in the YGS57 (rga1Δ rga2Δ bem3Δ) strain. Nomarski optics was used to examine cell morphology and an α-Cdc3 antibody was used to reveal septins. Localization patterns are shown for round, peanut, and fingered cells. See Table 4 for quantitation.

FIG. 4.

Haploid invasive growth phenotypes of strains lacking the Cdc42 GAPs. (A) Cells of the Σ1278b background were grown for 4 days at 30°C on synthetic medium. Plates were washed with water and rubbed with a wet finger. (B) Single-cell invasive growth assay. Equal concentrations of wild-type (wt), rga1, rga2, and bem3 cells were spread onto SCD (+Glu) or SC (−Glu) medium, incubated for 16 h at 25°C, and photographed at 100× by placing a coverslip directly on the agar medium. All images were taken at the same scale. Bar, 30 μm.

FIG. 5.

Temperature-dependant growth of strains lacking a PAK kinase and one of the Cdc42 GAPs. (A) Aliquots of 10 μl of a 1:100 dilution of IDY22 (cla4Δ), YGS80 (rga1Δ cla4Δ), YGS81 (rga2Δ cla4Δ), or YGS82 (bem3Δ cla4Δ) mid-log phase liquid cultures were spotted onto rich plates and incubated at the indicated temperatures. (B) Aliquots of 10 μl of a 1:1,000 and a 1:10,000 dilution of YGS 286 (ste20Δ), YGS 351 (rga1Δ ste20Δ), YGS 352 (rga2Δ ste20Δ), and YGS 353 (bem3Δ ste20Δ) mid-log phase liquid cultures were spotted onto rich plates and incubated at the indicated temperatures.

FIG. 6.

Effects of deletion or overexpression of Cdc42 GAPs on two-hybrid interaction between Ste20 and activated versions of Cdc42. (A) The interaction of Ste20 residues 1 to 565 fused to the Gal4 DBD (pSL2682) with three versions of the Gal4 transcription activation domain—GAD itself and GAD fused either to Cdc42^G12V,C188S (pGS38) or to Cdc42^Q61L,C188S (pGS39)—in four separate strains were compared: PJ69-4A, YGS156 (rga1Δ), YGS157 (rga2Δ), and YGS158 (bem3Δ). Expression of the lacZ reporter in three separate isolates was measured. (B) The same two-hybrid interactions investigated above were compared for strain PJ69-4A transformed with four different plasmids: YEp352, pGS40 (YEp352-RGA1), pGS41 (YEp352-RGA2), and pGS42 (YEp352-BEM3). The expression of the lacZ reporter was measured for three separate isolates in four independent trials. The combined normalized data were graphed.