title: Genetic and biochemical studies of a mutant Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase, nmt72pLeu99-->Pro, that produces temperature-sensitive myristic acid auxotrophy.
abstract: Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that transfers myristate from CoA to the amino-terminal glycine residue of at least 12 cellular proteins. Its reaction mechanism is Ordered Bi Bi with myristoyl-CoA binding occurring before binding of nascent polypeptides and release of CoA preceding release of the myristoylprotein product. nmt1-72 is a temperature-sensitive allele, identified by Stone et al. (Stone, D. E., Cole, G. M., Lopes, M. B., Goebl, M., and Reed, S. I. (1991) Genes & Dev. 5, 1969-1981) that causes arrest in the G1 phase of the cell cycle due to reduced acylation of Gpa1p. We have recovered this mutant allele and determined that it contains a single point mutation resulting in a Leu99 (CTA) to Pro (CCA) substitution. Addition of > or = 500 microM myristate but not palmitate to synthetic or rich media rescues the growth arrest caused by nmt1-72 at 37-39 degrees C, consistent with the observation that purified nmt72p has reduced affinity for myristoyl-CoA and that exogenous myristate but not palmitate increases cellular myristoyl-CoA pools. Metabolic labeling studies in S. cerevisiae and co-expression of nmt72p with several protein substrates of Nmt1p in Escherichia coli indicate that the Leu99-->Pro substitution causes a reduction in the acylation of some but not all protein substrates. Since formation of a myristoyl-CoA.Nmt1p complex appears to be required for synthesis/formation of a peptide binding site, these defects in acylation appear to arise either because Leu99 is a component of the enzyme's functionally distinguishable myristoyl-CoA and peptide recognition sites or because Pro99 alters the interaction between myristoyl-CoA and enzyme in a way that precludes formation of a normal peptide binding site. The reduction in affinity for myristoyl-CoA produced by Leu99-->Pro in nmt72p is less than that produced by the Gly451-->Asp mutation in nmt181p, which also produces temperature-sensitive myristic acid auxotrophy. Isogenic, haploid strains containing NMT1, nmt1-72, and nmt1-181 do not manifest any obvious differences in steady state levels of the acyltransferases during growth at permissive temperatures or in the biosynthesis of long chain saturated acyl-CoAs. The spectrum of cellular N-myristoylproteins whose level of acylation is affected by nmt1-72 and nmt1-181 is distinct.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: D R
authors.initials: DR
authors.lastname: Johnson
authors.forename: R J
authors.initials: RJ
authors.lastname: Duronio
authors.forename: C A
authors.initials: CA
authors.lastname: Langner
authors.forename: D A
authors.initials: DA
authors.lastname: Rudnick
authors.forename: J I
authors.initials: JI
authors.lastname: Gordon
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D011392:Proline; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D013816:Thermodynamics
chemical_list: D009227:Myristic Acids; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019814:Myristic Acid; D011392:Proline; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D007930:Leucine