Robert J. Duronio

Protein N-myristoylation.

Protein N-myristoylation refers to the covalent attachment of myristic acid, a 14-carbon saturated fatty acid (C14:0), to the N-terminal glycine of proteins. Linkage occurs via an amide bond and takes place as proteins are being synthesized. N-myristoylproteins have varied intracellular destinations, and are involved in myriad cellular functions ranging from signal transduction to protein and vesicular trafficking. N-myristoylproteins are encountered in members of all kingdoms of the eukaryotic domain (Protist, Fungi, Plant, and Animal), but are not produced by members of Bacteria or Archaea. MyristoylCoA: protein N-myristoyltransferase (Nmt), E.C. 2.3.1.97, a member of the GCN5 acetyltransferase (GNAT) superfamily, is responsible for catalyzing the transfer of myristate from myristoylCoA to proteins. While the acylCoA substrate specificity of Nmt has been highly conserved during evolution, its peptide substrate specificities have diverged among eukaryotes.

Reconstitution of protein N-myristoylation in Escherichia coli

A dual-plasmid expression system that coexpresses Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) and various known mammalian N-myristoylproteins in Escherichia coli is described. Coexpression of these proteins results in efficient N-myristoylation in the bacterium, which has no endogenous NMT activity. N-Myristoylation in E. coli requires that the protein substrate have a Gly2 residue, and is specific for 14-carbon fatty acids as in eukaryotes. The system is useful for (i) analyzing NMT structure/activity relationships, (ii) examining the determinants required for efficient N-myristoylation in vivo, and (iii) directly assessing the biological significance of this modification of specific eukaryotic proteins by purifying the corresponding nonmyristoylated and N-myristoylated recombinant forms from E. coli.