Singularity in budding: a role for the evolutionarily conserved small GTPase Cdc42p

Abstract

The budding yeast Saccharomyces cerevisiae initiates polarized growth or budding once per cell cycle at a specific time of the cell cycle and at a specific location on the cell surface. Little is known about the molecular nature of the temporal and spatial regulatory mechanisms. It is also unclear what factors, if any, among the numerous proteins required to make a bud are involved in the determination of budding frequency. Here we describe a class of cdc42 mutants that produce multiple buds at random locations on the cell surface within one nuclear cycle. The critical mutation responsible for this phenotype affects amino acid residue 60, which is located in a domain required for GTP binding and hydrolysis. This mutation bypasses the requirement for the essential guanine-nucleotide-exchange factor Cdc24p, suggesting that the alteration at residue 60 makes Cdc42p hyperactive, which was confirmed biochemically. This result also suggests that the only essential function of Cdc24p is to activate Cdc42p. Together, these data suggest that the temporal and spatial regulation of polarized growth converges at the level of Cdc42p and that the activity of Cdc42p determines the budding frequency.

Fig 1.

cdc42-22 cells produce multiple buds during one mass-doubling time. (A) Cells of YEF1963 (cdc42-22) carrying pRS316-CDC3-GFP were grown on a SC-Ura plate at 24°C for 16 h and then observed by DIC and fluorescence microscopy. (B) Time-lapse analysis of the budding process of YEF473A (WT) and YEF1963. All images in the same panel are at the same magnification.

Fig 2.

cdc42-22 cells produce multiple simultaneously growing buds within one nuclear cycle. (A) Cells of JPC40 (cdc42-22, TUB1:GFP) were fixed with ice-cold 70% ethanol and stained for F-actin and DNA. Microtubules were visualized with a GFP-tagged α-tubulin. (B) Cells of YEF1963 (cdc42-22) were pulse-labeled with FITC-Con A for 10 min and then released into fresh medium for 60 min to monitor the deposition of new cell wall materials (dark zones on the cell surface). (C) Cells of YEF1963 and YEF473A (WT) carrying pSM217-GIC2-GFP were fixed with ice-cold 70% ethanol and stained for DNA.

Fig 3.

cdc42-22 cells bud randomly. Cells of YEF473A (WT) and YEF1963 (cdc42-22) were stained for bud scars with Calcofluor.

Fig 4.

A single amino acid change in a GTP-binding and hydrolysis domain is responsible for the hyperactivity of cdc42-22 allele. (A) Cell lysates from YEF473A (WT), JPC250 [WT+CDC42 (OE)], YEF1963 (cdc42-22), and other cdc42Δ strains carrying different alleles of CDC42 (strains JPC152–156) were probed with antibodies against Cdc42p and, as a control, the mitochondrial outer membrane protein Isp42p. (B) Cell lysates from YEF473A (WT), YEF1963 (cdc42-22), and JPC241 (cdc42^G60D) were assayed for the active portion of Cdc42p by GST-PBD pull downs.

Fig 5.

A single change of Cdc42p at glycine 60 allows the bypass of its GEF, Cdc24p. (A) Temperature-sensitive growth of cdc24Δ strains carrying a hyperactive cdc42 allele. Strains YEF473A (WT), YEF1963 (cdc42-22), JPC160 (cdc24Δ, pRS314-CDC42-22), and JPC162 (cdc24Δ, pRS314-CDC42-G60A) were streaked onto YPD plates and incubated for 2–4 days at 24°C and 37°C, respectively. (B) Western-blot analysis of cell lysates from YEF473A (WT), YEF1963 (cdc42-22), JCY403 [WT+CDC24 (OE)], and other cdc24Δ strains carrying different alleles of CDC42 (strains JPC160–164).

Fig 6.

A model for the role of Cdc42p in budding. (A) In WT cells, Cdc42p is subjected to temporal (CDK/cyclins) and spatial (bud-site selection) controls. The activity of Cdc42p is adequate for a single round of budding. (B) In cdc42^G60X cells, hyperactive Cdc42p overrides the temporal and spatial controls, and the activity of Cdc42p is sufficient for multiple rounds of budding in a single cell cycle time. The Cdc42p GTPase module includes Cdc42p itself and its immediate regulators such as GEF and GAPs.