Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation.

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.

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.