Abstract
Covalent attachment of ubiquitin is well-known to target proteins for degradation. Here, mass spectrometry was used to identify the site of ubiquitination in Gpa1, the G protein alpha subunit in yeast Saccharomyces cerevisiae. The modified residue is located at Lys165 within the alpha-helical domain of Galpha, a region of unknown function. Substitution of Lys165 with Arg (Gpa1(K165R)) results in a substantial decrease in ubiquitination. In addition, yeast expressing the Gpa1(K165R) mutant are moderately resistant to pheromone in growth inhibition assays-a phenotype consistent with enhanced Galpha signaling activity. These findings indicate that the alpha-helical domain may serve to regulate the turnover of Gpa1.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Substitution / genetics
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Arginine / genetics
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Chromatography, Liquid
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Cysteine Endopeptidases / metabolism
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GTP-Binding Protein alpha Subunits*
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GTP-Binding Protein alpha Subunits, Gq-G11
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Growth Inhibitors / pharmacology
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Heterotrimeric GTP-Binding Proteins / genetics
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Heterotrimeric GTP-Binding Proteins / isolation & purification*
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Heterotrimeric GTP-Binding Proteins / metabolism
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Lysine / genetics
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Multienzyme Complexes / metabolism
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Pheromones / physiology
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Proteasome Endopeptidase Complex
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / isolation & purification*
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Saccharomyces cerevisiae Proteins / metabolism
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Signal Transduction / genetics
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Spectrometry, Mass, Electrospray Ionization / methods
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Ubiquitin / metabolism*
Substances
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GTP-Binding Protein alpha Subunits
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Growth Inhibitors
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Multienzyme Complexes
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Pheromones
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Saccharomyces cerevisiae Proteins
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Ubiquitin
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Arginine
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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GPA1 protein, S cerevisiae
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GTP-Binding Protein alpha Subunits, Gq-G11
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Heterotrimeric GTP-Binding Proteins
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Lysine