Cok S J, Martin C G, Gordon J I
Department of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine,660 South Euclid Avenue, St Louis, MO 63110, USA.
Nucleic Acids Res. 1998 Jun 15;26(12):2865-72. doi: 10.1093/nar/26.12.2865.
Inositol regulates transcription of Saccharomyces cerevisiae genes required for de novo synthesis of acylCoAs and phospholipids. Removal of inositol results in transcriptional activation by heterodimeric complexes of two bHLH proteins, Ino2p and Ino4p. In the presence of inositol, transcription is repressed by Opi1p. MyristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme whose activity is influenced by cellular myristoylCoA pool size and availability. nmt451Dp contains a Gly451-->Asp substitution that produces temperature-dependent reductions in affinity for myristoylCoA and associated reductions in acylation of cellular N-myristoylproteins. The conditional lethality produced by nmt1-451D is rescued at temperatures up to 33 degreesC by withdrawal of inositol. We tested the hypothesis that N-myristoylproteins function to regulate INO2, INO4 and/or OPI1 transcription, thereby affecting the expression of inositol-sensitive genes that influence myristoylCoA metabolism. The effect of nmt1-451D on INO2 , INO4 and OPI1 promoter activities was examined by introducing episomes, containing their 5' non-transcribed domains linked to reporters, into isogenic NMT1 and nmt1-451D cells. The activity of INO2 is significantly higher, INO4 significantly lower and OPI1 unaffected in nmt1-451D cells, both in the presence and absence of inositol. These changes are associated with a net increase in expression of some inositol target genes, including FAS1 . FAS1 encodes one of the subunits of the fatty acid synthase complex that catalyzes de novo acylCoA (including myristoylCoA) biosynthesis. Augmented expression of FAS1 overcomes the kinetic defects in nmt451Dp. FAS1 expression is Ino2p-dependent in NMT1 cells at 24-33 degreesC. In contrast, FAS1 expression becomes Ino2p-independent in nmt1-451D cells at temperatures where efficient acylation of cellular N-myristoylproteins is jeopardized. The ability to maintain expression of FAS1 in nmt1-451Dino2 Delta cells suggests the existence of another transcription factor, or factors, whose expression/activity is inversely related to overall levels of cellular protein N-myristoy-lation. This factor is not functionally identical to Ino2p since other inositol-responsive genes (e.g. CHO1 ) maintain INO2 -dependent expression in nmt1-451D cells.
肌醇可调节酿酒酵母中从头合成酰基辅酶A和磷脂所需基因的转录。去除肌醇会导致由两种bHLH蛋白Ino2p和Ino4p组成的异二聚体复合物激活转录。在有肌醇存在的情况下，转录受到Opi1p的抑制。肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶（Nmt1p）是一种必需酶，其活性受细胞内肉豆蔻酰辅酶A库大小和可用性的影响。nmt451Dp含有一个Gly451→Asp替换，该替换导致对肉豆蔻酰辅酶A的亲和力随温度降低，并使细胞内N-肉豆蔻酰化蛋白质的酰化作用相应降低。nmt1-451D产生的条件致死性在高达33℃的温度下通过去除肌醇得以挽救。我们测试了以下假设：N-肉豆蔻酰化蛋白质的功能是调节INO2、INO4和/或OPI1的转录，从而影响影响肉豆蔻酰辅酶A代谢的肌醇敏感基因的表达。通过将含有与报告基因相连的5'非转录结构域的附加体导入同基因的NMT1和nmt1-451D细胞，研究了nmt1-451D对INO2、INO4和OPI1启动子活性的影响。在有和没有肌醇的情况下，nmt1-451D细胞中INO2的活性显著更高，INO4的活性显著更低，而OPI1的活性不受影响。这些变化与一些肌醇靶基因（包括FAS1）表达的净增加相关。FAS1编码脂肪酸合酶复合物的一个亚基，该复合物催化从头合成酰基辅酶A（包括肉豆蔻酰辅酶A）。FAS1表达的增加克服了nmt451Dp中的动力学缺陷。在24-33℃时，NMT1细胞中FAS1的表达依赖于Ino2p。相反，在细胞内N-肉豆蔻酰化蛋白质有效酰化受到损害的温度下，nmt1-451D细胞中FAS1的表达变得不依赖于Ino2p。在nmt1-451Dino2Δ细胞中维持FAS1表达的能力表明存在另一种转录因子，其表达/活性与细胞内蛋白质N-肉豆蔻酰化的总体水平呈负相关。该因子在功能上与Ino2p不同，因为其他肌醇反应基因（如CHO1）在nmt1-451D细胞中维持依赖于INO2的表达。
