Johnson D R, Cok S J, Feldmann H, Gordon J I
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10158-62. doi: 10.1073/pnas.91.21.10158.
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.
几种重要的酿酒酵母蛋白质需要肉豆蔻酸共价结合到其氨基末端甘氨酸上才能具有生物活性。蛋白质N-肉豆蔻酰化由肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶Nmt1p催化。nmt1-181编码一种具有Gly451→Asp替换的突变酶。nmt181p对肉豆蔻酰辅酶A的亲和力降低，在≥30℃时导致蛋白质N-肉豆蔻酰化出现全局性缺陷。nmt1-181导致细胞在转移至≥30℃后1小时内的细胞周期各个阶段生长停滞，并在8小时内致死。生长停滞表型和活力丧失不需要交配途径的成分，并且与裂解敏感性相关，这可能与两种蛋白磷酸酶Ppz1p和Ppz2p的肉豆蔻酰化不足有关。在30℃时，通过向培养基中添加肉豆蔻酸或山梨醇或去除肌苷可以挽救生长。在37℃时，通过过表达nmt1-181p或Nmt1p或向培养基中添加肉豆蔻酸可以挽救细胞。在37℃观察到的肉豆蔻酸营养缺陷型和致死性的高拷贝抑制子筛选仅得到NMT1，而当在30℃进行筛选时，获得了六个与肉豆蔻酰化缺陷无关的抑制子（SMD1-6）。鉴定了三个SMD基因座的蛋白质产物：（i）cdc39-δ1.7p，其可反式激活NMT1；（ii）Fas1p，脂肪酸合成酶复合体β亚基，可激活FAS2的启动子并增加Gpa1p的肉豆蔻酰化；（iii）Pho5p，这种酵母产生的主要分泌酸性磷酸酶。当酵母在缺磷培养基中生长时，PHO5通常被诱导。从培养基中去除无机磷酸盐也可在30℃挽救nmt1-181细胞。PHO5抑制nmt1-181的机制似乎至少部分涉及FAS2转录的激活以及对FAS1表达的后续影响。细胞N-肉豆蔻酰转移酶活性和分泌酸性磷酸酶活性之间存在负相关。这些观察结果为将磷酸盐代谢与肉豆蔻酰辅酶A合成和蛋白质N-肉豆蔻酰化的调节联系起来提供了一种潜在机制。
