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 and 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 ≥500 μM myristate but not palmitate to synthetic or rich media rescues the growth arrest caused by nmt1-72 at 37-39°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. This is evidenced by: (i) differences in pattern of incorporation of exogenous [3H]myristate into N-myristoylproteins, (ii) the fact that disruption of STE4 is epistatic to nmt1-72 but not to nmt1-181, (iii) the lack of effect of overexpression of Gpa1p on growth of isogenic haploid nmt1-181 strains, (iv) differences in the degree of growth suppression produced by overexpression of Arf1p, and (v) differences in their abilities to acylate a panel of four substrates of Nmt1p in the E. coli co- expression system. Together, these findings suggest that nmt1-72 and nmt1- 181 strains may be useful for identifying essential cellular N- myristoylproteins as well as gene products that affect myristoyl-CoA metabolism and/or the delivery of this substrate to Nmt1p.
|Number of pages||12|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|