Direct identification of a G protein ubiquitination site by mass spectrometry.
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.
Marotti
Louis A
LA
Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Newitt
Rick
R
Wang
Yuqi
Y
Aebersold
Ruedi
R
Dohlman
Henrik G
HG
D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D006131:Growth Inhibitors / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D009097:Multienzyme Complexes / Q000378:metabolism; D010675:Pheromones / Q000502:physiology; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism*
D044385:GTP-Binding Protein alpha Subunits; D006131:Growth Inhibitors; D009097:Multienzyme Complexes; D010675:Pheromones; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D001120:Arginine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D008239:Lysine