Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins. Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates. To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene. A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1 alleles encoding enzymes with reduced activity. nmt1 alleles were identified that produced temperature-sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn102, Ala202, Cys217, Ser328, Val395, Asn404, Leu420, and Asn426. Ala202 --> Thr, Cys217 --> Arg, Ser328 --> Pro, Asn404 --> Tyr, and Asn426 --> Ile produced the most severe ts phenotype. Their effects on the functional properties of the enzyme's myristoyl-CoA and peptide binding sites were defined by purifying each mutant from Escherichia coli and conducting in vitro kinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p). None of the substitutions affect the enzyme's acyl chain length selectivity. When compared with wild type Nmt1p, Cys217 --> Arg produces 3- and 6-fold increases in Ki for SC-58272 at 24 and 37 degrees C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site. Asn426 --> Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold). Ala202 --> Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the Ki for S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the Ki for SC-58272 at 24 and 37 degrees C. An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions. The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to Arf1p.
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| Gene | Phenotype | Experiment Type | Mutant Information | Strain Background | Chemical | Details |
|---|---|---|---|---|---|---|
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-202T A202T (nucleotide G604A); peptide binding site altered; myristoyl-CoA binding site decreased; reduction in specific activity | Other | Temperature: elevated temperature, 30 °C | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-102T N102T (nucleotide A305C); reduction in specific activity | Other | Temperature: elevated temperature, 40 °C | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-395D V395D (nucleotide T1184A); reduction in specific activity | Other | Temperature: elevated temperature, 40 °C | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-420S L420S (nucleotide T1259C); reduction in specific activity | Other | Temperature: elevated temperature, 40 °C Details: reduced steady state levels of the mutated protein at elevated temperature | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-328P S328P (nucleotide T982C); reduction in specific activity | Other | Temperature: elevated temperature, 35 °C Details: reduced steady state levels of the mutated protein at all temperatures tested | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-426I N426I (nucleotide A1277T); myristoyl-CoA binding site mutated; reduction in specific activity | Other | Temperature: elevated temperature, 35 °C | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-404Y N404Y (nucleotide A1210T); temperature dependent changes in myristoyl-CoA and peptide binding; reduction in specific activity | Other | Temperature: elevated temperature, 37 °C Details: reduced steady state levels of the mutated protein at all temperatures tested | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-217R C217R (nucleotide T649C); peptide binding site altered; reduction in specific activity | Other | Temperature: elevated temperature, 39 °C | |
| NMT1 | heat sensitivity: increased | classical genetics | conditional Allele: nmt1-181 G451D; Km for myristoyl-CoA increased 10-fold and catalytic activity reduced 200-fold at 37 deg C | Other | Temperature: elevated temperature, 40 °C | |
| NMT1 | protein/peptide modification: decreased Reporter: Arf1p | classical genetics | conditional Allele: nmt1-404Y N404Y (nucleotide A1210T); temperature dependent changes in myristoyl-CoA and peptide binding; reduction in specific activity | Other | Temperature: permissive temperature, 24 °C Details: reduction in N-myristoylation of Arf1p with ~60% acylated at 24 deg C and <50% at 37 deg C but this can be increased to >95% with the addition of myristate |