Altered membrane association of p60v-src and a murine 63-kDa N-myristoyl protein after incorporation of an oxygen-substituted analog of myristic acid.
A number of viral and cellular proteins contain covalently bound lipid. In a subset of these acyl proteins, the 14-carbon saturated fatty acid myristic acid is attached through an amide linkage to an NH2-terminal glycine residue. Myristoyl-CoA:protein N-myristoyltransferase (NMT) transfers the myristoyl moiety from myristoyl-CoA to these nascent proteins and is highly selective for fatty acid chain length. We have found that 10-(propoxy)decanoyl-CoA (11-oxymyristoyl-CoA), an analog of myristic acid with reduced hydrophobicity, acts as a substrate for NMT both in vitro and in vivo. Comparison of the in vitro kinetic properties of a number of synthetic octapeptide substrates of NMT using myristoyl-CoA or 11-oxymyristoyl-CoA indicated that there is an interaction between the acyl-CoA and peptide binding sites of this acyltransferase. Peptide catalytic efficiency with 11-oxymyristoyl-CoA was reduced relative to that with myristoyl-CoA, but the extent of the reduction varied widely among the octapeptides tested. These in vitro data accurately predicted that only a subset of myristoyl proteins synthesized in Saccharomyces cerevisiae and a murine myocyte-like cell line (BC3H1) would incorporate 11-oxy[3H]myristate. Substitution of the myristoyl moiety by the 11-oxymyristoyl moiety does not significantly affect the membrane association of most N-myristoyl proteins. However, for the tyrosine kinase p60v-src and a 63-kDa N-myristoyl protein in BC3H1 cells, analog incorporation results in marked redistribution from the membrane to the cytosolic fraction. These studies demonstrate the utility of heteroatom-containing analogs for analysis of the role of myristate in acyl protein targeting. The sequence-specific nature of analog incorporation and the protein-specific effects on membrane association suggests that these compounds may represent a useful class of antiviral and antitumor agents.
Heuckeroth
R O
RO
Department of Biochemistry, Washington University School of Medicine, Saint Louis, MO 63110.
Gordon
J I
JI
D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009227:Myristic Acids / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src); D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D014316:Tritium
D005656:Fungal Proteins; D008565:Membrane Proteins; D009227:Myristic Acids; D012191:Retroviridae Proteins; D014316:Tritium; C060254:10-propoxydecanoic acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)