Amino Acid Residue Penultimate to the Amino-terminal Gly Residue Strongly Affects Two Cotranslational Protein Modifications, N-Myristoylation andN-Acetylation

Abstract

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 Å 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.

Rapid detection, discovery, and identification of post‐translationally myristoylated proteins during apoptosis using a bio‐orthogonal azidomyristate analog

“…Like other previously identified post-translationally myristoylated proteins, the proteins we identified are kinases, proapoptotic proteins, or regulators of the cytoskeleton structure (9,10,31). Like ctPAK2, caspase cleavage of PKCε results in the loss of the N-terminal regulatory domain to generate a constitutively active C-terminal kinase domain (35).…”

Topogenesis of Two Transmembrane Type K+ Channels, Kir 2.1 and KcsA

“…Plasmids pBV2G,R3A-TNF and pBV2G,R3D-TNF (previously designated, respectively, as pBR3A-TNF and pBR3D-TNF) were constructed as described previously (27). Plasmid pBKir 2.1-TNF was constructed by PCR using pBDpro-TNF as template and the primers, 5Ј-ATATGGATCCATGGGCAG-TGTGCGAACCAACCGCTACAGCGACAAGCCTGTAGCC-3Ј and 5Ј-G-CCGGGATCCTAGGGCGAATTGGGTACC-3Ј.…”

“…The amino acid sequence of the N terminus of Kir 2.1 (MGSVRTNR) fits to the N-myristoylation consensus motif, MGXXX(S/T)XX (27,30,31). Protein N-myristoylation results from the cotranslational addition of myristic acid, a 14-carbon saturated fatty acid, to the Gly residue at the extreme N terminus after removal of the initiating Met by methionine aminopeptidase (32).…”

Machine Learning Prediction of Amino Acid Patterns in Protein N-myristoylation

“…As shown in Fig. 3, when Ser is located at position 6, 11 amino acid residues (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) are permitted locating at position 3 to direct efficient protein N-myristoylation [2,3]. Most of these 11 amino acids have a rule that the radius of gyration of residue is smaller than 1.80Å.…”

“…In order to determine the amino-terminal sequence requirements for protein N-myristoylation, their sequences have been examined [2,3]. Most of methods used by researchers are those that predict patterns for N-myristoylation by biological experimentations based on their knowledge.…”