RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation.

Abstract

Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family. These two genes, RAS] and RAS2, constitute an essential gene family since spores with disruptions ofboth genes fail to grow. We report here that strains containing RAS2 disruptions have three distinct phenotypes. First, they fail to grow efficiently on nonfermentable carbon sources. Like their mammalian counterparts, the yeast RAS proteins bind GTP (7, 8) and, at least in the case of RAS1, have an intrinsic GTPase activity similar to that found in p21 (9). This activity is decreased or absent in RAS1 proteins containing missense mutations at amino acid positions analogous to transforming mutations in p21 (9). Functional similarities also exist between p21 and yeast RAS. The normal mammalian Harvey ras gene (10) or the viral Harvey ras (11) can substitute for both of the normal RAS genes in yeast and a modified RAS] gene can transform NIH-3T3 cells (11).Given the functional and biochemical similarities between mammalian and yeast RAS proteins, it is likely that knowledge about RAS function in yeast may have direct implications forRAS function in mammals. We report here that yeast strains lacking RAS2 function have a defect in gluconeogenic growth, accumulate excessive levels of storage carbohydrates, and sporulate prematurely. We have identified mutations in three additional genes that suppress the gluconeogenic defect. Two of these mutations allow strains without RAS to grow. MATERIALS AND METHODSYeast Strains. TX2-530.1.1C (a leu2 ura3 trpl lysi lys2 his3 ras2-530) (4) was backcrossed to MCY638 (a his4-539 lys2-801 ura3-52). MCY638 and MCY317 (a ade2-101 his4-539), derived from strain S288C, were kindly provided by Marian Carlson. Meiotic segregants of EG81, the second backcross to MCY638, were used for quantitative measurements and revertant analysis. Although backcrosses were performed to show linkage between specific phenotypes and ras2, mendelian segregation patterns for the gluconeogenic defect and glycogen hyperaccumulation were clearly observed in original transformed diploid TX2-530. Specific strains used include EG81-40A (a ura3 his4 lys2), EG81-40B (a ras2-530 leu2 ura3 his4 lys2 lysi), EG81-40C (a ras2-530 leu2 ura3 his4 lys2 his3 lysl?), EG81-40D (a ura3 his4 lys2 his3 lysl?), , and 112-699 [a leu2 ura3 can1-100 ade2-1 his3 ras2-699 (His+)].The gluconeogenic revertants described below were derived from EG81-22B. Revertants were crossed to EG72-16C (a ras2-530 leu2 trpl lys2) and all subsequent crosses between the strains containing sra mutations were done with meiotic progeny from these crosses. EG73-14D (a rasl-545 leu2 ura3 lysi his3) and EG87-2B (a rasl-545 ura3 his4 lys2) were used to test if the sra mutations bypass RAS. XC0262485 [a rasl485 (Trp') stell'3 leu2 his4-580 his3 trpl] has been described elsewhere (7).Media and Genetic Techniques. Unless otherwise stated, yeast cells were grown on 1% yeast extract and 2% peptone (YEP medium) containing 2% of the designated carbon sourc...

SDC25, a dispensable Ras guanine nucleotide exchange factor of Saccharomyces cerevisiae differs from CDC25 by its regulation.

“…As an example, it has been shown that RASI is expressed during growth on fermentable carbon sources and is repressed on nonfermentable carbon sources, whereas RAS2 is expressed under both conditions (this situation leads RAS2 to be essential on nonfermentable carbon sources) (Tatchell et al, 1985;Breviario et al, 1986). In this report, we found that SDC25 is not transcribed during growth on glucose; this might explain why CDC25 is essential under these conditions.…”

ASC1/RAS2 Suppresses the Growth Defect on Glycerol Caused by the atp1–2 Mutation in the YeastSaccharomyces cerevisiae

“…One phenotype of different alleles of ras2 is the failure to grow on a non-fermentable carbon source, although a firm characterization of the mechanism of this remains open. RAS2 activates adenylyl cyclase, followed by activation of cAMPdependent protein kinases (42)(43)(44). The presence of a cAMPdependent protein kinase on the inner membrane of mitochondria has been reported (45,46).…”

The yeast MCK1 gene encodes a protein kinase homolog that activates early meiotic gene expression.

“…Entry into the sporulation pathway requires starvation and expression of the al and a2 products, which determine the a/a cell type. The starvation signal is transmitted through decreased cAMPdependent protein kinase activity, and through a cAMPindependent pathway{s} (Matsumoto et al 1983;Tatchell et al 1984Tatchell et al , 1985Olempska-Beer and Freese 1987;Cameron et al 1988;Gibbs and Marshall 1989}. al and et2 are products of the MATa and MATa alleles, respectively, and only cells expressing both {typically MATa/MAToL diploids) are sporulation proficient {Her-skowitz 1988}.…”