Johnson D R, Knoll L J, Rowley N, Gordon J I
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1994 Jul 8;269(27):18037-46.
NMT1 is an essential Saccharomyces cerevisiae gene which encodes myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). Nmt1p transfers myristate (C14:0), from myristoyl-CoA to the amino-terminal Gly residue of several essential cellular proteins. Little information is available about how myristoyl-CoA metabolism is regulated in eukaryotic cells. We have isolated and characterized three unlinked Fatty Acid Activation genes from S. cerevisiae, FAA1, FAA2, and FAA3. In vitro biochemical assays reveal that the myristoyl-CoA synthetase activity of purified Faa2p is approximately equal to that of Faa1p, and two orders of magnitude greater than that of Faa3p. Analysis of NMT1 strains containing faa1, faa2, and/or faa3 null alleles indicates that Faa1p, Faa2p, and Faa3p are not essential for vegetative growth when de novo acyl-CoA synthesis by fatty acid synthetase (Fas) is active. S. cerevisiae strains containing nmt1-181 exhibit temperature-sensitive growth arrest and myristic acid auxotrophy due to the reduced affinity of its mutant protein product (nmtGly451-->Asp) for myristoyl-CoA. Comparison of the growth characteristics of isogenic NMT1 and nmt1-181 strains with all possible combinations of faa1, faa2, and faa3 null alleles, in the presence or absence of an active Fas complex, indicates that (i) Faa1p is responsible for activation of imported fatty acids to their CoA derivatives; (ii) Faa2p and Faa3p are able to access endogenous but not imported fatty acid substrates; (iii) nmt181p requires myristoyl-CoA production from both Fas and Faas for cells to remain viable at nonpermissive temperatures; (iv) Faa2p is unique among the three Faas in its ability, when overproduced, to partially rescue growth of a nmt1-181 strain at nonpermissive temperatures on yeast/peptone/dextrose (YPD) media without C14:0 supplementation; (v) acyl-CoAs produced by Faa1p, Faa2p, or Faa3p are not specifically targeted for beta-oxidation; and (vi) the ability of NMT1, faa1 delta, faa2 delta, faa3 delta strains to remain viable in the absence of an active Fas complex on YPD plus C14:0, or on media that contains fatty acids as the sole carbon source, suggests that S. cerevisiae contains other acyl-CoA synthetases which can activate imported fatty acids.
NMT1是酿酒酵母中的一个必需基因，它编码肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶（Nmt1p）。Nmt1p将肉豆蔻酸（C14:0）从肉豆蔻酰辅酶A转移到几种必需细胞蛋白的氨基末端甘氨酸残基上。关于真核细胞中肉豆蔻酰辅酶A代谢如何被调控的信息很少。我们从酿酒酵母中分离并鉴定了三个不连锁的脂肪酸激活基因，即FAA1、FAA2和FAA3。体外生化分析表明，纯化的Faa2p的肉豆蔻酰辅酶A合成酶活性与Faa1p大致相当，比Faa3p高两个数量级。对含有faa1、faa2和/或faa3无效等位基因的NMT1菌株的分析表明，当脂肪酸合成酶（Fas）进行从头酰基辅酶A合成活跃时，Faa1p、Faa2p和Faa3p对于营养生长不是必需的。含有nmt1 - 181的酿酒酵母菌株表现出温度敏感的生长停滞和肉豆蔻酸营养缺陷，这是由于其突变蛋白产物（nmtGly451→Asp）对肉豆蔻酰辅酶A的亲和力降低所致。将同基因的NMT1和nmt1 - 181菌株与faa1、faa2和faa3无效等位基因的所有可能组合的生长特性进行比较，在有或没有活跃的Fas复合物存在的情况下，表明：（i）Faa1p负责将导入的脂肪酸激活为其辅酶A衍生物；（ii）Faa2p和Faa3p能够利用内源性而非导入的脂肪酸底物；（iii）nmt181p需要Fas和Faa产生的肉豆蔻酰辅酶A，以便细胞在非允许温度下保持存活；（iv）在不添加C14:0的酵母/蛋白胨/葡萄糖（YPD）培养基上，当过量表达时，Faa2p在三个Faa中具有独特能力，能够部分挽救nmt1 - 181菌株在非允许温度下的生长；（v）Faa1p、Faa2p或Faa3p产生的酰基辅酶A并非专门用于β-氧化；（vi）NMT1、faa1Δ、faa2Δ、faa3Δ菌株在没有活跃的Fas复合物存在时，在YPD加C14:0或含有脂肪酸作为唯一碳源的培养基上仍能存活，这表明酿酒酵母含有其他能够激活导入脂肪酸的酰基辅酶A合成酶。
