Met-Gly-Cys motif from G-protein alpha subunit cannot direct palmitoylation when fused to heterologous protein.
To determine whether the N-terminal Met-Gly-Cys motif from G-protein alpha subunit can direct palmitoylation of protein, we have generated heterologous fusion proteins containing the first 10 amino acids of Gi1 alpha and Gs alpha using tumor necrosis factor as a model protein and determined their ability to incorporate palmitate using in vitro and in vivo expression systems. DNA sequences coding for the N-terminal 10 amino acids of Gi1 alpha and Gs alpha were fused to the 5'-end of the cDNA coding for the mature domain of tumor necrosis factor (TNF) to give Gi1 alpha-TNF and Gs alpha-TNF cDNA. In vitro translation of the mRNA coding for the Gi1 alpha-TNF cDNA gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. In contrast, no incorporation of fatty acid was detected for Gs alpha-TNF. Baculovirus expression of the Gi1 alpha-TNF cDNA in Sf-9 cells gave rise to an N-myristoylated but not palmitoylated fusion TNF. This myristoylation was inhibited by replacement of Gly-2 with Ala but not Cys-3 with Ala, indicating the acylation reaction is entirely dependent on the N-myristoylation signal (Met-Gly-X-X-X-Ser) and Cys-3 is not involved. As is the case with in vitro translation, no incorporation of fatty acid was detected for Gs alpha-TNF. These results indicated that unlike the myristoylation signal Met-Gly-X-X-X-Ser/Thr/Cys, the Met-Gly-Cys motif found in G-protein alpha subunits itself is not sufficient to direct palmitoylation even if Gly-2 is myristoylated after removal of initiating Met. Thus, another structural determinant is implicated in this modification.
Utsumi
T
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Tou
E
E
Takemura
D
D
Ishisaka
R
R
Yabuki
M
M
Iwata
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005998:Glycine; D046911:Macromolecular Substances; D008715:Methionine; D019814:Myristic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D019814:Myristic Acid; D019308:Palmitic Acid; D008715:Methionine; D019205:GTP-Binding Protein alpha Subunits, Gs; D003545:Cysteine; D005998:Glycine