Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.
Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.
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nmtl-181（酿酒酵母肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶基因的条件致死等位基因）的抑制子揭示了参与调节蛋白质N-肉豆蔻酰化的蛋白质。
DOI：
10.1073/pnas.91.21.10158
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发表时间：
1994
影响因子：
11.1
通讯作者：
Gordon,JI
中科院分区：
文献类型：
--
作者：
Johnson,DR;Cok,SJ;Feldmann,H;Gordon,JI
几种必需的酿酒酵母蛋白需要肉豆蔻酸酯共价结合到其氨基末端甘氨酸以获得生物活性。蛋白N-肉豆蔻酰化由肉豆蔻酰辅酶A催化：蛋白N-肉豆蔻酰转移酶，Nmt 1 p。nmt 1 -181编码具有Gly 451->Asp取代的突变酶。nmt 181 p对肉豆蔻酰辅酶A的亲和力降低，并在≥ 30 ℃时产生蛋白N-肉豆蔻酰化的整体缺陷。nmt 1 -181导致细胞在转移至≥ 30 ℃后1小时内在细胞周期的各个阶段生长停滞，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组分，并与裂解敏感性相关，这可能与两种蛋白磷酸酶Ppz 1 p和Ppz 2 p的豆蔻酰化不足有关。在30 ℃时，通过向培养基中加入肉豆蔻酸酯或山梨醇或除去肌苷，可以挽救生长。细胞可以在37 ℃下通过过表达nmt 1 - 181 p或Nmt 1 p或通过向培养基中添加肉豆蔻酸酯来拯救。在37 ℃下观察到的肉豆蔻酸营养缺陷和致死性的高拷贝抑制子的选择仅产生NMT 1，而在30 ℃下进行筛选时获得了6个未连接的肉豆蔻酰化缺陷（SMD 1 -6）抑制子。鉴定了三个SMD基因座的蛋白产物：（i）cdc 39-delta 1.7p，其反式激活NMT 1;（ii）Fas 1 p，脂肪酸合成酶复合物的β亚基，其激活FAS 2的启动子并增加Gpa 1 p的肉豆蔻酰化;和（iii）Pho 5 p，由该酵母产生的主要分泌型酸性磷酸酶。当酵母在磷酸盐耗尽的培养基中生长时，PHO 5通常被诱导。从培养基中去除无机磷酸盐也可以在30 ℃下拯救nmt 1 -181细胞。PHO 5抑制nmt 1 -181的机制似乎至少部分涉及FAS 2转录的激活和对FAS 1表达的影响。细胞N-肉豆蔻酰转移酶和分泌的酸性磷酸酶活性之间存在反比关系。这些观察结果提供了一个潜在的机制，耦合磷酸盐代谢与肉豆蔻酰辅酶A的合成和蛋白质N-肉豆蔻酰化的调节。
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.