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 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.
Highlights
A number of eukaryotic cellular proteins are found to be covalently modified with the 14-carbon saturated fatty acid, myristic acid [1,2,3,4,5,6]
Amino Acid at Position 3 in the N-Myristoylation Consensus Motif Strongly Affects Protein N-Myristoylation and N-Acetylation—To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, and to reveal the difference in the N-terminal sequence requirement for protein N-myristoylation and N-acetylation, the N-terminal 9 residues of the mature domain of tumor necrosis factor (TNF) including the initiating Met were changed to the N-myristoylation consensus motif, and the susceptibility to cotranslational protein N-myristoylation and Nacetylation was evaluated by an in vitro translation system
Since ⌬pro-TNF, a mature domain of TNF in which the initiating Met was introduced at the N terminus, has Met and Ser residues at positions 1 and 6, respectively, Val at position 2 was replaced with Gly to obtain V2G-TNF, in which the N-terminal 9 residues were adapted to the N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X (Fig. 1)
Summary
Materials—Restriction endonucleases, DNA-modifying enzymes, RNase inhibitor, and Taq DNA polymerase were purchased from Takara Shuzo (Kyoto, Japan). Plasmid pBOVA, which contains the full-length chicken ovalbumin cDNA was constructed by using PCR In this case, pET-22b-OVA served as a template, and two oligonucleotides (OVA-N and OVA-C) served as primers (Table I). The cDNA coding for OVA60-TNF in which the N-terminal 60 residues of ovalbumin were linked to the N terminus of the mature domain of TNF was constructed by using PCR For this procedure, pBOVA served as a template, and two oligonucleotides (OVA-N and OVA-60) served as primers (Table I). The cDNAs coding for Arf6-TNF and hippocalcin-TNF, in which the N-terminal 10 residues of ⌬pro-TNF were replaced with those of Arf or hippocalcin, were constructed by using PCR For this procedure, pB⌬pro-TNF served as a template, and two oligonucleotides (Arf plus B1 and HC plus B1, respectively) served as primers (Table I). Radioactivity on the thin layer plate was made visible by spraying with En3Hance (PerkinElmer Life Sciences)
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