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.
Utsumi
T
T
Department of Biological Chemistry and Department of Veterinary Medicine, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. utsumi@agr.yamaguchi-u.ac.jp
Sato
M
M
Nakano
K
K
Takemura
D
D
Iwata
H
H
Ishisaka
R
R
D000107:Acetylation; D020816:Amino Acid Motifs; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009227:Myristic Acids / Q000378:metabolism*; D010047:Ovalbumin / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D018076:DNA, Complementary; D009227:Myristic Acids; D014409:Tumor Necrosis Factor-alpha; D010047:Ovalbumin; D005998:Glycine