Vertical-scanning mutagenesis of amino acids in a model N-myristoylation motif reveals the major amino-terminal sequence requirements for protein N-myristoylation.
In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N-myristoylation.
Utsumi
Toshihiko
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan. utsumi@yamaguchi-u.ac.jp
Nakano
Kengo
K
Funakoshi
Takeshi
T
Kayano
Yoshiyuki
Y
Nakao
Sayaka
S
Sakurai
Nagisa
N
Iwata
Hiroyuki
H
Ishisaka
Rumi
R
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics*; D000818:Animals; D019556:COS Cells; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009227:Myristic Acids / Q000378:metabolism*; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014162:Transfection; D014316:Tritium; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000596:Amino Acids; D017931:DNA Primers; D018076:DNA, Complementary; D009227:Myristic Acids; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014316:Tritium