Isolation and Characterization of WHI 3 , a Size-Control Gene of Saccharomyces cerevisiae

WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae. The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase. WHI3 seems to be a dose-dependent inhibitor of Start. Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function. CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor. Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function. MOST eukaryotic cells become committed to cell known to be present is Cln3, which is expressed reladivision at a control point called the restriction tively constitutively through the cell cycle. Cln3 forms point, the commitment point, or, in yeasts, Start (Harta complex with Cdc28, and this protein kinase complex well et al. 1974; Pardee 1989; Zetterberg et al. 1995; somehow, perhaps in a size-dependent way, activates the Neufeld and Edgar 1998). In some organisms, includtranscription factors SBF and MBF (Tyers et al. 1993; ing the yeasts Saccharomyces cerevisiae and SchizosaccharoKoch and Nasmyth 1994). These in turn cause the myces pombe, growth to a critical cell volume is a prerequitranscription of z200 genes in late G1 phase, including site for Start (Johnston et al. 1977; Mitchison and CLN1 and CLN2 (Spellman et al. 1998). Cln1-Cdc28 Nurse 1985; Nash et al. 1988). Mutations that alter the and Cln2-Cdc28 then presumably phosphorylate other critical volume required for Start have defined imporsubstrates such as Sic1, and these phosphorylation tant cell cycle control genes, including the G1 cyclin events constitute Start. gene CLN3 (originally called WHI1; Carter and SudMany environmental signals regulating Start do so by bery 1980; Sudbery et al. 1980; Cross 1988; Nash et influencing the synthesis or activity of the G1 cyclins. al. 1988). Of particular interest are genes that affect Cln1, Cln2, and Cln3 are each unstable proteins with critical volume in a dose-dependent way. For instance, half-lives of z10 min (Schneider et al. 1998), that are two copies of CLN3 cause a smaller critical volume than encoded by unstable mRNAs. This instability makes Cln one copy, and higher doses can decrease volume still levels very responsive to changing environmental condifurther (Nash et al. 1988). One interpretation of dose tions. Thus, a drop in the rate of protein synthesis dependence is that the amount of gene product is being quickly reduces Cln levels, and this is probably importitrated against some other metric (e.g., nuclear volume, tant for the cell’s ability to arrest in G1 in response to amount of DNA) and the ratio is sensed by the cell. starvation. Nutrients are known to affect cell size, and Critical cell volume is not the only requirement for this is partly due to signaling through the RAS-cAMP Start. For instance, cells can pass through Start only if pathway to Cln synthesis (Tokiwa et al. 1994). Mating they are growing and making protein. However, a wide pheromones arrest cells in G1 phase because signaling range of protein synthesis rates is acceptable for Start, through the mating pathway shuts off the synthesis of whereas the critical volume is relatively well defined. Cln1 and Cln2, and perhaps their activity as well (GartThus it appears that volume is measured by the cell as ner et al. 1998). one requirement for Start. There is still no clear idea of how critical size is meaIn S. cerevisiae, Start depends on the Cdc28 protein sured by the cell. That is, why do cells undertake Start kinase and the G1 cyclins Cln1, Cln2, and Cln3 (Richat some cell sizes but not at others? What happens durardson et al. 1989). Cdc28 becomes activated by associing the long G1 phase of a slowly growing daughter cell ating with a cyclin. In small G1 cells, the only cyclin that eventually allows Start? We have taken a genetic approach to this problem by seeking new mutations that affect critical cell size. Mutations that allow cells to Corresponding author: Bruce Futcher, Department of Molecular Geundertake Start at smaller critical cell sizes will yield netics and Microbiology, State University of New York, Stony Brook, NY 11794-5222. E-mail: bfutcher@ms.cc. sunysb.edu strains that have small average cell sizes—a Whi phenoGenetics 157: 1469–1480 (April 2001) 1470 R. S. Nash, T. Volpe and B. Futcher