title: The role of palmitoylation of the guanine nucleotide binding protein G11 alpha in defining interaction with the plasma membrane.
abstract: Mutations of Cys-9 to serine, Cys-10 to serine and a combination of both alterations were produced in a cDNA encoding murine G11 alpha to potentially interfere with the ability of the expressed polypeptides to act as substrates for post-translational palmitoylation. Each of these mutants and the wild-type protein were expressed in simian COS-1 cells. Mutation of either cysteine-9 or cysteine-10 decreased the degree of palmitoylation of the protein by some 80% compared with the wild-type, while the double mutant totally failed to incorporate [3H]palmitate. By contrast, in all transfections the endogenously expressed simian G11 alpha incorporated [3H]palmitate to similar levels. Particulate and cytoplasmic fractions from these cells were subjected to SDS/PAGE under conditions which allow resolution of primate and rodent forms of G11 alpha. Immunoblotting of these fractions demonstrated that in all cases the endogenously expressed simian G11 alpha was exclusively associated with the particulate fraction, as was the transfected and expressed wild-type murine G11 alpha. By contrast, each of the mutated forms of murine G11 alpha displayed a distribution in which approx. 70% of the expressed protein was present in the particulate fraction and 30% in the supernatant. To examine the conformation of the particulate expressed forms of murine G11 alpha, these fractions were treated with various concentrations of sodium cholate and immunoblots were subsequently performed on the solubilized and remaining particulate proteins. Whereas essentially all of the endogenous simian G11 alpha was solubilized by treatment with 1% (w/v) sodium cholate and some 50% with 0.32% cholate, expressed wild-type murine G11 alpha was more recalcitrant to solubilization. However, that fraction of wild-type murine G11 alpha which was solubilized behaved identically to the endogenous simian G11 alpha on Superose-12 gel-exclusion chromatography. The particulate fraction of the C9S/C10S double mutant of murine G11 alpha was highly resistant to solubilization by sodium cholate, whereas the particulate fractions of the two single cysteine to serine mutants were intermediate to the wild-type and double mutant in their ability to be solubilized by this detergent. These data demonstrate that the palmitoylation status of the cysteine residues at positions 9 and 10 in murine G11 alpha plays a central role in defining membrane association of this G-protein and indicate that much of the particulate fraction of the expressed palmitoylation-resistant mutants is likely to represent non-functional rather than correctly folded protein.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
authors.forename: J F
authors.initials: JF
authors.lastname: McCallum
authors.forename: A
authors.initials: A
authors.lastname: Wise
authors.forename: M A
authors.initials: MA
authors.lastname: Grassie
authors.forename: A I
authors.initials: AI
authors.lastname: Magee
authors.forename: F
authors.initials: F
authors.lastname: Guzzi
authors.forename: M
authors.initials: M
authors.lastname: Parenti
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D003545:Cysteine; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014644:Genetic Variation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D010169:Palmitic Acids; D011994:Recombinant Proteins; D019308:Palmitic Acid; D012694:Serine; D019204:GTP-Binding Proteins; D003545:Cysteine