Multiple regulatory mechanisms control the expression of the RAS1 and RAS2 genes of Saccharomyces cerevisiae.

Breviario, Diego; Hinnebusch, Alan G.; Dhar, Ravi

doi:10.1002/j.1460-2075.1988.tb03012.x

Cited by 43 publications

(33 citation statements)

References 40 publications

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“…Previously, Ras1p and Ras2p were thought to be the main regulators of cAMP in S. cerevisiae, demonstrating a role for Ras1p only in the early exponential growth phase (18). However, we have shown here that deletion of both RAS2 and GPA2 reveals that the remaining activity of Ras1p is not sufficient to induce normal cAMP levels and, thus, not sufficient to ensure wild-type-like growth.…”

Section: Discussioncontrasting

confidence: 54%

Gpa2p, a G-protein α-Subunit, Regulates Growth and Pseudohyphal Development in Saccharomyces cerevisiae via a cAMP-dependent Mechanism

Kübler¹,

Mösch²,

Rupp³

et al. 1997

Journal of Biological Chemistry

191

184

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The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein ␣-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein ␣-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation.In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein ␣-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein ␣-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein ␣-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.When shifted to nitrogen starvation conditions, diploid cells of the yeast Saccharomyces cerevisiae switch from normal vegetative growth to filamentous growth (1-3). This switch in developmental patterning is regulated by intracellular signaling pathways.Many signaling molecules have been identified that control filamentous growth. These include the genes encoding the protein kinase Ste20p (a homologue of mammalian p65PAK protein kinases), Ste11p (a MEKK or MAPK kinase kinase), 1 Ste7p (a MEK or MAPK kinase), and the transcription factor Ste12p (4). Furthermore, during nitrogen starvation the Ras2 protein, in addition to activating adenylyl cyclase (5, 6), induces filamentous growth by stimulating the MAPK pathway (7). This pathway also regulates pheromone responsiveness via the evolutionary conserved Cdc42p/Ste20p/MAPK module (7). These observations suggest that in response to a nutritional signal, the MAPK pathway is activated by a cascade of small G-proteins, similar to the activation of the Jun N-terminal kinase pathway in mammalian cells (8).Although the pheromone response pathway and the pathway regulating filamentous growth share a common MAPK module, the upstream regulators of each pathway seem to be specific to a given pathway. For example, neither the G-protein ␣-subunit (Gpa1p) nor the G␤/␥-protein subunits (Ste4p/Ste18p) of the heterotrimeric G-protein that regulate...

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Section: Discussioncontrasting

confidence: 54%

Gpa2p, a G-protein α-Subunit, Regulates Growth and Pseudohyphal Development in Saccharomyces cerevisiae via a cAMP-dependent Mechanism

Kübler¹,

Mösch²,

Rupp³

et al. 1997

Journal of Biological Chemistry

191

184

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“…The apparent differential requirement of Erf2 depending on the RAS gene being expressed could arise from differences in the level of Ras activity in ras2⌬ and ras1⌬ cells or perhaps a functional difference between the two Ras proteins. RAS1 and RAS2 are differentially expressed depending on growth conditions and the position in the growth curve (10,11). However, we did not observe a difference in the steady-state levels of Ras1 and Ras2 proteins in our strains (Fig.…”

Section: Bmentioning

confidence: 52%

Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

Bartels

Mitchell

Dong

et al. 1999

Molecular and Cellular Biology

167

200

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Ras proteins are small membrane-associated GTP binding proteins that cycle between active (GTP-bound) and inactive (GDP-bound) states to regulate cell growth and differentiation. In Saccharomyces cerevisiae, two Ras proteins (Ras1 and Ras2) affect such diverse processes as vegetative growth, sporulation, carbon source utilization, stress response, and pseudohyphal growth (12,25,45,61,64). Ras-dependent growth requires plasma membrane localization, which in turn depends on a series of posttranslational modifications that occur on a carboxyl-terminal CaaX box (C is cysteine, a is any aliphatic residue, X is the carboxy-terminal residue) (15,20,21,57). The first step is the farnesylation of the CaaX box cysteine by a soluble, heterodimer farnesyl protein transferase encoded by the RAM1 and RAM2 genes in yeast (34,39,53). The aaX sequence is removed by one of two endoplasmic reticulum (ER)-associated proteases encoded by RCE1 and AFC1/STE24 (9, 58). The newly exposed cysteinyl ␣-carboxyl is then methyl esterified by the product of STE14, an integral membrane protein which also colocalizes with the ER in yeast and mammalian cells (16,18,22,54,56).The mature form of Ras is localized primarily on the cytoplasmic surface of the plasma membrane. Mutations in either the CaaX box or the genes encoding the posttranslational modification enzymes cause a reduction in Ras plasma membrane localization and a corresponding reduction in the ability of Ras to support growth (15, 57). However, the mechanism by which prenylation and subsequent posttranslational modifications direct Ras proteins to the plasma membrane is not known. It is clear that prenylation alone is not sufficient for efficient plasma membrane targeting of Ras. Yeast mutants that fail to carry out the palmitoylation step have reduced amounts of Ras protein at the plasma membrane and increased resistance to heat shock in the presence of activated Ras2(V19) alleles (6). Palmitoylation, unlike the CaaX processing steps, is reversible, making it a likely regulatory step. Interestingly, oncogenic forms of Ras are also rendered nontransforming by mutating the palmitoylation sites (69). Unfortunately, palmitoylation is the least well understood step in the Ras posttranslational modification pathway. A major obstacle has been the failure to identify a protein palmitoyltransferase, although reports of partial purification of palmitoyltransferase activities have appeared (3,19,37). The matter is further complicated by the fact that protein palmitoylation can occur nonenzymatically; however, the reaction rate is considerably lower than that observed in vivo (1,23,66). The issue is unlikely to be resolved until there is a better understanding of the requirements for and biological consequences of palmitoylation in vivo.The multistep nature of Ras modification suggests that subcellular targeting may be an ordered process involving distinct intracellular membrane compartments. In support of this idea is the recent demonstration that the -aaX proteases (Afc1 and Rce1) and methylt...

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“…For example, the RAS2 transcript declines to nondetectable levels after the diauxic shift, but the rate of synthesis of its protein, Ras2p, is constant throughout the culture cycle (7,8). Additionally, accumulation of BCYJ mRNA is comparable in stationary phase and exponential phase, but accumulation of Bcylp is approximately 10-fold higher in stationary phase than in exponential phase (49,52).…”

Section: Od600mentioning

confidence: 99%

Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae

1994

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We are interested in characterizing the process of entry into and the maintenance of the stationary phase. To identify proteins that are induced during growth to stationary phase, we examined protein synthesis in long-term stationary-phase cultures using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).Although the total rate of protein synthesis declined when growth ceased after the postdiauxic phase, the pattern of proteins synthesized remained similar throughout the experimental period (28 days), except at the diauxic shift. At the diauxic shift most proteins detectable by 2D-PAGE undergo a transient reduction in their relative rate of synthesis that ends when cells resume growth during the postdiauxic phase. We conclude from this that the transient repression of protein synthesis at the diauxic shift is not directly associated with stationary-phase arrest. A number of proteins that are synthesized after exponential phase have been identified by 2D-PAGE. These proteins could be divided into three temporal classes depending upon when their synthesis became detectable. One postexponential protein, designated p35, was induced later than all other proteins, and its relative rate of synthesis increased throughout stationary phase. Unlike most postexponential proteins, p35 was not regulated by heat shock or glucose repression. We also observed that a direct correlation between steady-state mRNA accumulation and protein synthesis for another postexponential protein (Ssa3p) or two closely related constitutive proteins (Ssalp and Ssa2p) did not exist. We conclude from this result that synthesis of proteins in stationary phase is regulated by mechanisms other than the control of steady-state mRNA accumulation.

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Multiple regulatory mechanisms control the expression of the RAS1 and RAS2 genes of Saccharomyces cerevisiae.

Cited by 43 publications

References 40 publications

Gpa2p, a G-protein α-Subunit, Regulates Growth and Pseudohyphal Development in Saccharomyces cerevisiae via a cAMP-dependent Mechanism

Gpa2p, a G-protein α-Subunit, Regulates Growth and Pseudohyphal Development in Saccharomyces cerevisiae via a cAMP-dependent Mechanism

Erf2, a Novel Gene Product That Affects the Localization and Palmitoylation of Ras2 in Saccharomyces cerevisiae

Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae

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