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 pro tein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated mod el protein. Subsequently, the susceptibility of these mutants to protein N- myristoylation was evaluated by metabolic labeling in an in vitro translati on 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 c otranslational protein modifications, N-myristoylation and N-acetylation; 1 0 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, lie, Gin, and His) with a radius of gyration smaller than 1.80 Angstrom directed N-myristoylation, t wo negatively charged residues (Asp and Glu) directed N-acetylation, and tw o amino acids (Gly and Met) directed heterogeneous modification with both N -myristoylation and N-acetylation, The amino acid requirements at this posi tion for the two modifications were dramatically changed when Ser at positi on 6 in the consensus motif was replaced with Ale. Thus, the amino acid res idue penultimate to the N-terminal Gly residue strongly affected two cotran slational protein modifications, N-myristoylation and N-acetylation, and th e amino acid requirements at this position for these two modifications were significantly affected by downstream residues.