Genetic and biochemical studies of a mutant Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase, nmt72pLeu99-->Pro, that produces temperature-sensitive myristic acid auxotrophy

J Biol Chem. 1993 Jan 5;268(1):483-94.

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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acyltransferases / genetics*
  • Acyltransferases / isolation & purification
  • Acyltransferases / metabolism*
  • Alleles
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Genes, Fungal*
  • Genotype
  • Kinetics
  • Leucine*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Myristic Acid
  • Myristic Acids / metabolism*
  • Oligodeoxyribonucleotides
  • Open Reading Frames
  • Proline
  • Promoter Regions, Genetic
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / growth & development
  • Temperature
  • Thermodynamics

Substances

  • Myristic Acids
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Myristic Acid
  • Proline
  • Acyltransferases
  • glycylpeptide N-tetradecanoyltransferase
  • Leucine