Met-Gly-Cys Motif from G-Protein α Subunit Cannot Direct Palmitoylation When Fused to Heterologous Protein

To determine whether the N-terminal Met-Gly-Cys motif from G-protein α subunit can direct palmitoylation of protein, we have generated heterologous fusion proteins containing the first 10 amino acids of Gi1α and Gsα using tumor necrosis factor as a model protein and determined their ability to incorporate palmitate usingin vitroandin vivoexpression systems. DNA sequences coding for the N-terminal 10 amino acids of Gi1α and Gsα were fused to the 5′-end of the cDNA coding for the mature domain of tumor necrosis factor (TNF) to give Gi1α-TNF and Gsα-TNF cDNA.In vitrotranslation of the mRNA coding for the Gi1α-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 Gsα-TNF. Baculovirus expression of the Gi1α-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 within vitrotranslation, no incorporation of fatty acid was detected for Gsα-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 α 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.