Functional analysis of protein N-myristoylation: metabolic labeling studies using three oxygen-substituted analogs of myristic acid and cultured mammalian cells provide evidence for protein-sequence-specific incorporation and analog-specific redistribution.

Abstract

Covalent attachment of myristic acid (C14:0) to the NH2-terminal glycine residue of a number of cellular, viral, and oncogene-encoded proteins is essential for full expression of their biological function. Substitution of oxygen for methylene groups in this fatty acid does not produce a significant change in chain length or stereochemistry but does result in a reduction in hydrophobicity. These heteroatom-containing analogs serve as alternative substrates for mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97) and offer the opportunity to explore structure/function relationships of myristate in N-myristoyl proteins. We have synthesized three tritiated analogs of myristate with oxygen substituted for methylene groups at C6, C11, and C13. Metabolic labeling studies were performed with these compounds and (i) a murine myocyte cell line (BC3H1), (ii) a rat fibroblast cell that produces p60v-src (3Xsrc), or (iii) NIH 3T3 cells that have been engineered to express a fusion protein consisting of an 11-residue myristoylation signal from the Rasheed sarcoma virus (RaSV) gag protein linked to c-Ha-ras with a Cys----Ser-186 mutation. This latter mutation prevents isoprenylation and palmitoylation of ras. Two-dimensional gel electrophoresis of membrane and soluble fractions prepared from cell lysates revealed different patterns of incorporation of the analogs into cellular N-myristoyl proteins (i.e., protein-sequence-specific incorporation). In addition, proteins were identified that underwent redistribution from membrane to soluble fractions after incorporating one but not another analog (analog-specific redistribution). Comparable studies using the model RaSV-ras chimeric protein also demonstrated analog-specific differences in incorporation, varying from approximately 25% of the total RaSV-ras chimeric protein with 5-octyloxypentanoate to greater than 50% with 12-methoxydodecanoate. Modification by this latter compound was so extensive that the amount of membrane-associated N-myristoylated protein was decreased. Incorporation of each of the analogs caused a dramatic redistribution to the soluble fraction, comparable to that seen when myristoylation was completely blocked by mutating the protein's site of myristate attachment (glycine) to an alanine residue. The demonstration that these analogs differ in the extent to which they are incorporated and in their ability to cause redistribution of any single protein suggests that they may also have sufficient selectivity to be of potential therapeutic value.