metglycys motif from gprotein alpha subunit cannot direct palmitoylation when fused to heterologous protein to determine whether the nterminal metglycys motif from gprotein 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 nterminal 10 amino acids of gi1 alpha and gs alpha were fused to the 5end of the cdna coding for the mature domain of tumor necrosis factor tnf to give gi1 alphatnf and gs alphatnf cdna in vitro translation of the mrna coding for the gi1 alphatnf cdna gave rise to an nmyristoylated fusion tnf with a molecular mass of 18 kda as determined by the incorporation of 3hmyristic acid and by immunoprecipitation with antitnf antibody in contrast no incorporation of fatty acid was detected for gs alphatnf baculovirus expression of the gi1 alphatnf cdna in sf9 cells gave rise to an nmyristoylated but not palmitoylated fusion tnf this myristoylation was inhibited by replacement of gly2 with ala but not cys3 with ala indicating the acylation reaction is entirely dependent on the nmyristoylation signal metglyxxxser and cys3 is not involved as is the case with in vitro translation no incorporation of fatty acid was detected for gs alphatnf these results indicated that unlike the myristoylation signal metglyxxxserthrcys the metglycys motif found in gprotein alpha subunits itself is not sufficient to direct palmitoylation even if gly2 is myristoylated after removal of initiating met thus another structural determinant is implicated in this modification