GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae.

Miyajima, Ikuko; Arai, Kohei; Matsumoto, Kunihiro

doi:10.1128/mcb.9.6.2289

Cited by 58 publications

(73 citation statements)

References 34 publications

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“…4), as was also observed by others for mutations at this amino acid [Miyajima et al 1989;Stone and Reed 1990}. The a-factor spot results in a small area of growth inhibition of wild-type cells and a large, clear zone of growth arrest of an sst2 mutant, which is defective in desensitization to phero- (Chan and Otte 1982;Dietzel and Kurjan 1987b).…”

Section: Glycine To Valine Substitution Results In Multiple Effectsmentioning

confidence: 58%

“…11. This mutant showed multiple phenotypes, some of which differ from results of Miyajima et al (1989; see Discussionl. The a scgl :: LEU2 [pTRP1-MATa YEp352-scgl w15°] strain could lose pTRP1-MATa (Table 1); the resulting strain showed normal growth but was sterile.…”

Section: Glycine To Valine Substitution Results In Multiple Effectsmentioning

confidence: 83%

“…2), indicating that basal activation of the pathway is less extreme than in the complete absence of Scgl. Miyajima et al (1989) did not see a growth defect associated with this mutation (see below).…”

Section: Gxxxxgk Domain Glycine To Valine Substitution Has Multiple Ementioning

confidence: 99%

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Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Kurjan

Hirsch²,

Dietzel³

1991

Genes Dev.

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Several domains of guanine nucleotide-binding proteins are conserved and form the guanine nucleotide-binding pocket. Mutations in these domains in EF-Tu, ras, and Gas have been shown to result in informative phenotypes. We made several analogous changes in SCG1, which encodes the ,x subunit of the G protein involved in pheromone response in yeast. The scgl L~sass and scgl An°a91 mutations resulted in s e v e r e growth and cell morphology defects; this phenotype is similar to the null phenotype and results from constitutive activation of the pheromone response pathway. On the basis of the model for the action of the yeast G protein, the effect of these mutations is consistent with the effect of analogous mutations in ras, which result in a transforming phenotype. The SCG1 A~°a22 mutation resulted in pheromone response and mating defects. This effect is similar to the effect of the analogous Gas mutation, which results in a defect in stimulation of adenylate cyclase. The scgl was° mutation, which is analogous to the transforming mutation ras v"n2, resulted in multiple effects, including defects in growth, cell morphology, and mating. Some of our results and interpretations are different from previously published results of others for the same mutation in SCG1; specifically, our gene replacement of this mutation resulted in high basal activation of the pheromone response pathway, consistent with a GTPase defect, which was not seen previously with scgl V~a5° on a low-copy plasmid. Implications of these phenotypes are discussed.

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Section: Glycine To Valine Substitution Results In Multiple Effectsmentioning

confidence: 58%

Section: Glycine To Valine Substitution Results In Multiple Effectsmentioning

confidence: 83%

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Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Kurjan

Hirsch²,

Dietzel³

1991

Genes Dev.

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“…The pheromone response pathway is exquisitely sensitive to changes in Gpa1p expression (Cole et al, 1990;Kang et al, 1990). Studies of other GPA1 mutations have revealed different phenotypes when expressed on a centromere-based plasmid instead of the chromosomal locus (Miyajima et al, 1989;Kurjan et al, 1991). We found that haploid cells expressing the mutant allele as the sole copy of GPA1 were viable.…”

Section: Gpa1p Is Dually Myristoylatedmentioning

confidence: 70%

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

Manahan

Patnana

Blumer

et al. 2000

MBoC

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To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein ␥ subunit (Ste18p) is unusual among G ␥ subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation-or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G ␥ subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G ␤␥ after receptor-stimulated release from G ␣ . The G protein ␣ subunit (Gpa1p) is tandemly modified at its N terminus with amide-and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway. INTRODUCTIONLipid modifications anchor heterotrimeric guanine nucleotide-binding proteins (G proteins) to the inner leaflet of the plasma membrane. G protein ␣ subunits are fatty acylated with amide-linked myristate, thioester-linked palmitate, or both. G protein ␥ subunits are prenylated with either farnesyl or geranylgeranyl moieties through stable thioether linkages. Prenylation of ␥ subunits and myristoylation of ␣ subunits are essential for the function of G proteins. These modifications promote plasma membrane association and facilitate high-affinity protein-protein interactions (reviewed in Wedegaertner et al., 1995). The functional consequences of thioacylation are less well understood. Thioacylation does not appear to be a major determinant of membrane avidity, at least in the presence of G ␤␥ subunits, but may play a role in targeting G ␣ specifically to the plasma membrane (Dunphy et al., 1996;Morales et al., 1998;Fishburn et al., 1999;Huang et al., 1999). In vitro studies have demonstrated the importance of thioester-linked lipid in mediating protein-protein interactions of G ␣ subunits. Thioacylation increases the affinity of G s␣ for G ␤␥ approximately fivefold (Iiri et al., 1996) and negatively regulates the interaction between regulators of G protein signaling and G ␣ subunits (Tu et al., 1997). Thus, thioacylation may impact protein-protein interactions, as well as the subcellular distribution of modified proteins.We have investigated thioacylation of G proteins in the genetically tractable organism Saccharomyces cerevisiae. The pheromone res...

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“…Other genetic observations suggested a direct interaction between SST2 and GPA1. First, certain mutations in the G␣ gene could bypass the requirement for a fully active SST2 gene (30,31). Second, the recovery-promoting effect of G␣ overexpression was more efficient if residual SST2 function was present (32,33).…”

Section: Establishing a Paradigm: Identification Of Yeast Sst2mentioning

confidence: 99%

RGS Proteins and Signaling by Heterotrimeric G Proteins

Dohlman

Thorner

1997

Journal of Biological Chemistry

490

370

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A ubiquitously employed mechanism for signal transduction involves ligand binding to a cell surface receptor coupled to a heterotrimeric guanine nucleotide-binding protein (G protein). Receptor activation stimulates nucleotide exchange and dissociation of the G protein, releasing the G␣ subunit in its GTP-bound state from the G␤␥ complex. The released subunits can stimulate a variety of target (effector) enzymes (1), thereby eliciting biochemical responses and changes in cellular physiology. Hundreds of G proteincoupled receptors have been identified (2, 3). These receptors share a common architecture containing seven membrane-spanning segments (4, 5). G proteins also comprise a superfamily that includes at least 17 distinct G␣ (6), 5 G␤, and 6 G␥ isoforms (1), allowing many combinatorial possibilities. Three-dimensional structures of several G␣ subunits and two different G␣␤␥ heterotrimers (7,8) have been determined, providing insights about how these molecular "switches" operate.How are the strength and duration of signaling adjusted to achieve an appropriate response? Attention in this regard has been devoted primarily to receptors, where phosphorylation by protein kinases (9) and receptor-binding proteins, like arrestins (10, 11), contribute to signal desensitization. However, additional proteins participate in signal attenuation at other levels, including phosducins (which act on G␤␥) (12) and recoverins (13,14). Here we focus on discovery of another superfamily of evolutionarily conserved proteins, dubbed RGS proteins, for "regulators of G protein signaling." RGS proteins act as negative regulators of G proteindependent signaling, at least in part, because they stimulate hydrolysis of the GTP bound to activated G␣ subunits. The Cycle of G Protein Activation and InactivationActivation of a G protein is initiated by agonist binding to a receptor, eliciting conformational change that is transmitted to the G protein, causing the G␣ subunit to release GDP and to bind GTP ( Fig. 1). GTP binding alters the conformation of three "switch" regions in G␣ that are its primary contact sites with G␤␥, promoting subunit dissociation (7,8). Guanine nucleotide exchange can occur spontaneously, but is accelerated by agonist-activated receptor and retarded by G␤␥ binding. Thus, the receptor acts as a GDS, 1 whereas G␤␥ acts as a GDI (15).Inactivation requires hydrolysis of the GTP bound to G␣, shifting the equilibrium in favor of subunit reassociation, preventing further signaling. Purified G␣ subunits display a measurable intrinsic rate of GTP hydrolysis, but the turnover number in vitro cannot account, in some systems, for the rate at which signaling is terminated in vivo (16,17). Other regulatory processes (such as those mediated by arrestin or phosducin) could be rate-limiting. Nonetheless, the importance of GTP hydrolysis provoked a search for factors that accelerate the GTPase activity of G␣ subunits, termed GAPs (for "GTPase-activating proteins") (Fig. 1). Effector enzymes can serve this role: phospholipase C␤ stimula...

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GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae.

Cited by 58 publications

References 34 publications

Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

RGS Proteins and Signaling by Heterotrimeric G Proteins

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GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae.

Cited by 58 publications

References 34 publications

Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway.

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

RGS Proteins and Signaling by Heterotrimeric G Proteins

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Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae