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 MG 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.