Several essential Saccharomyces cerevisiae proteins require myristate to be covalently bound to their amino-terminal glycine for biological activity. Protein N-myristoylation is catalyzed by myristoyl-CoA:protein N-myristoyl-transferase, Nmt1p. nmt1-181 encodes a mutant enzyme with a Gly451-->Asp substitution. nmt181p has a reduced affinity for myristoyl-CoA and produces global defects in protein N-myristoylation at > or = 30 degrees C. nmt1-181 results in growth arrest at various stages of the cell cycle within 1 hr after cells are shifted to > or = 30 degrees C and lethality within 8 hr. The growth-arrest phenotype and loss of viability do not require components of the mating pathway and are associated with lysis sensitivity that may be related to undermyristoylation of two protein phosphatases, Ppz1p and Ppz2p. Growth can be rescued at 30 degrees C by adding myristate or sorbitol to the medium or by removing inosine. Cells can be rescued at 37 degrees C by overexpressing nmt1-181p or Nmt1p or by adding myristate to the medium. Selection of high-copy suppressors of the myristate auxotrophy and lethality observed at 37 degrees C yielded only NMT1, whereas six unlinked suppressors of the myristoylation defect (SMD1-6) were obtained when the screen was conducted at 30 degrees C. The protein products of three SMD loci were identified: (i) cdc39-delta 1.7p, which transactivates NMT1; (ii) Fas1p, the beta subunit of the fatty acid synthetase complex, activates FAS2's promoter and increases myristoylation of Gpa1p; and (iii) Pho5p, the major secreted acid phosphatase produced by this yeast. PHO5 is normally induced when yeast are grown in phosphate-depleted medium. Removal of inorganic phosphate from the medium also rescues nmt1-181 cells at 30 degrees C. PHO5's mechanism of suppression of nmt1-181 appears to involve, at least in part, activation of FAS2 transcription and a resulting effect on FAS1 expression. There is an inverse relationship between cellular N-myristoyltransferase and secreted acid phosphatase activities. These observations provide a potential mechanism for coupling phosphate metabolism with the regulation of myristoyl-CoA synthesis and protein N-myristoylation.