Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase

Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT). NMT is absolutely specific for peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated. Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT. Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities. The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme.     

Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.     

11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase

The covalent attachment of myristic acid to the NH2-terminal glycine residue of proteins is catalyzed by the enzyme myristoyl CoA:protein N-myristoyltransferase (NMT). Using synthetic octapeptide substrates we have identified and characterized an NMT activity in wheat germ lysates used for cell-free translation of exogenous mRNAs. C-12 and C-14 fatty acids are efficiently transferred to the peptides by this plant NMT, but C-10 and C-16 fatty acids are not. Glycine is required as the NH2-terminal residue: peptides with an NH2-terminal alanine were not substrates. Peptides with proline, aspartic acid, or tyrosine residues adjacent to the NH2-terminal glycine were also not myristoylated. Serine in the fifth position reduced the peptide's Km up to 4000-fold. We have chemically synthesized a sulfur analogue of myristate, 11-(ethylthio)undecanoic acid. Its CoA ester is as good a substrate as myristoyl-CoA for both wheat germ and yeast NMT. Peptides linked to 11-(ethylthio)undecanoic acid are less hydrophobic than the corresponding myristoylpeptides. 11-(Ethylthio)-undecanoic acid may, therefore, help define the role of myristic acid in targeting of acyl proteins within cells.     

Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration

The muscle isoform of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.