N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase.

Stone, David E.; Cole, G M; Lopes, Miguel de Barros; Goebl, Mark G.; Reed, Steven I.

doi:10.1101/gad.5.11.1969

Cited by 85 publications

(77 citation statements)

References 58 publications

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“…Figure 5A, lane 1), consistent with thioacylation of the protein. As expected (Stone et al, 1991), [ 3 H]myristate was incorporated into Gpa1p ( Figure 5A, lane 1). To determine whether the cysteine residue at position 3 is required for thioacylation of Gpa1p, a mutant protein with an alanine substitution at this site (C3A Gpa1p) was expressed in yeast.…”

Section: A Prenylation and Thioacylation Motif Is Sufficient For Plassupporting

confidence: 82%

“…The pheromone response pathway in this species of yeast is regulated by a heterotrimeric G protein encoded by GPA1 (␣ subunit), STE4 (␤ subunit), and STE18 (␥ subunit) (reviewed in Sprague and Thorner, 1992;Leberer et al, 1997). In this pathway, ␤␥ propagates the signal from the receptor via the (Gpa1p) is myristoylated, and this modification is essential for viability of haploid yeast (Stone et al, 1991). A cysteine residue is found adjacent to the N-myristoylated glycine in Gpa1p, and palmitoylation of Gpa1p at this site has been reported .…”

Section: Introductionmentioning

confidence: 99%

“…The GPA1 gene product (Gpa1p) is myristoylated, and this modification is essential for viability of haploid yeast (Stone et al, 1991). A cysteine residue is found adjacent to the N-myristoylated glycine in Gpa1p, and palmitoylation of Gpa1p at this site has been reported .…”

Section: Introductionmentioning

confidence: 99%

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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

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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Section: A Prenylation and Thioacylation Motif Is Sufficient For Plassupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

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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

Manahan

Patnana

Blumer

et al. 2000

MBoC

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“…These deletions were expected to eliminate the putative N-terminal degradation signal in G␣/Gpa1. The initiator methionine of G␣ is normally excised to reveal a glycine residue (Gly 2 ) which is post-translationally attached to myristate (Stone et al 1991). Glycine is not a destabilizing residue under the criteria of the N-end rule (Varshavsky 1997) and Gly 2 is not required for targeting of G␣ ( Fig.…”

Section: Stability Of G␣ Mutantsmentioning

confidence: 99%

Sequence elements that contribute to the degradation of yeast Gα

Schauber¹,

Chen²,

Tongaonkar³

et al. 1998

Genes to Cells

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“…These proteins include Gpalp, the a subunit of a heterotrimeric G protein involved in the matingfactor signal transduction pathway (2,3), plus two functionally interchangeable ADP ribosylation factors, Arfip and Arf2p (4). Gpalp and the Arf proteins are essential and require myristate for expression of their biological functions.…”

mentioning

confidence: 99%

Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.

Johnson

Cok²,

Feldmann³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Several essential Saccharomyces cerevisiae proteins require myristate to be covalently bound to their amino-terminal glycine for biological activity. Protein N-myristoylation is catalyzed by myristoyl-CoA:protein N-myristoyltransferase, Nmtlp. nmtl-181 encodes a mutant enzyme with a Gly4s' -* Asp substitution. nmtl81p has a reduced affinity for myristoyl-CoA and produces global defects in protein N-myristoylation at .30°C. natl-181 results in growth arrest at various stages of the cell cycle within 1 hr after cells are shifted to .30°C and lethality within 8 hr. The growth-arrest phenotype and loss of viability do not require components ofthe mating pathway and are associated with lysis sensitivity that may be related to undermyristoylation of two protein phosphatases, Ppzlp and Ppz2p. Growth can be rescued at 300C by adding myristate or sorbitol to the medium or by removing inosine. Cells can be rescued at 37C by overexpressing nmtl181p or Nmtlp or by adding myristate to the medium. Selection of high-copy suppressors of the myristate auxotrophy and lethality observed at 3TC yielded only NMTI, whereas six unlinked suppressors of the myristoylation defect (SMDI-6) were obtained when the screen was conducted at 30'C. The protein products of three SMD loci were identified: (i) cdc39-A1.7p, which transactivates NMTI; (ii) Fasip, the ( subunit of the fatty acid synthetase complex, activates FAS2's promoter and increases myristoylation of Gpalp; and (iii) Pho5p, the major secreted acid phosphatase produced by this yeast. PHOS is normally induced when yeast are grown in phosphatedepleted medium. Removal of inorganic phosphate from the medium also rescues nmtl-181 cells at 30°C. PHOS's mechanism of suppression of nmtl-181 appears to involve, at least in part, activation of FAS2 transcription and a resulting effect on FASI expression. There is an inverse relationship between cellular N-myristoyltransferase and secreted acid phosphatase activities. These observations provide a potential mechanism for coupling phosphate metabolism with the regulation of myristoyl-CoA synthesis and protein N-myristoylation.

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N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase.

Cited by 85 publications

References 58 publications

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

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

Sequence elements that contribute to the degradation of yeast Gα

Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.

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N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase.

Cited by 85 publications

References 58 publications

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

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

Sequence elements that contribute to the degradation of yeast Gα

Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.

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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

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