A role for Saccharomyces cerevisiae fatty acid activation protein 4 in regulating protein N-myristoylation during entry into stationary phase
A role for Saccharomyces cerevisiae fatty acid activation protein 4 in regulating protein N-myristoylation during entry into stationary phase
复制标题
DOI：
10.1074/jbc.273.40.25864
复制
发表时间：
1998-10-02
影响因子：
4.8
通讯作者：
Gordon, JI
中科院分区：
文献类型：
--
作者：
Ashrafi, K;Farazi, TA;Gordon, JI
酿酒酵母含有四种已知的酰辅酶A合成酶(脂肪酸激活蛋白，Faaps)，Faa1p和Faa4p激活外源衍生的脂肪酸。酰辅酶A代谢在调节蛋白质N-肉豆蔻酰转移酶(Nmt1p)的重要酶--肉豆蔻酰辅酶A(Nmt1p)中起着关键作用。在这份报告中，我们研究了当细胞在营养缺乏(静止期)期间从富营养液中的生长过渡到生长停滞状态时，Faa1p和Faa4p是否在影响蛋白质N-肉豆蔻酰化方面具有不同的作用。10株同基因菌株的集落形成能力被定义为静止相停留时间的函数。这些菌株含有野生型或突变型NMTI等位基因，以及每种FAA的野生型或零等位基因。只有nmt突变体(nmt451Dp；对肉豆蔻酰-CoA的亲和力降低)和Faa4p的丢失组合才导致克隆形成单位(CFU)的显著损失。CFU的进行性百万倍减少与蛋白质N-肉豆蔻酰化缺陷有关，该缺陷首先出现在对数生长期，在二分生长期后恶化，在稳定期变得极端。Northern和Western印迹分析以及N-肉豆蔻酰基转移酶分析表明，NMT通常只在对数和二位期/后二位期存在，这表明在稳定期存在的N-肉豆蔻酰基蛋白是从这些早期阶段继承而来的。此外，FAA4是在关键的二生素/后二生素早期转换过程中唯一诱导的FAA。虽然nmt1-451D替换NMT1导致蛋白质N-肉豆蔻酸化的缺陷，但这些缺陷是温和的，并受到代偿反应的限制，包括nmt1-451D的增强表达和在对数生长期早熟诱导FAA4。从nmt1-451D细胞中丢失Faa4P严重损害了它们在进入固定相之前充分降解NMT底物的能力，因为其他FAAP都不能从功能上弥补它的缺失。为了确定在稳定期可能影响增殖潜能维持的Nmt1p底物，我们搜索了酵母基因组中已知的和可能的N-肉豆蔻基蛋白。在发现的基因中，有48个在NMT1细胞中被成功删除。去除以下九种底物中的任何一种都会产生与在nmt1-451Dfaa4 Delta细胞中观察到的类似的CFU损失：Arf1p、Arf2p、Sip2p、Van1p、Ptc2p、YBL049W(与Snf7p同源)、YJR114W、YKR007W和YMR077C。这些蛋白质提供了进一步确定在稳定期调节生存的分子机制的机会。
Saccharomyces cerevisiae contains four known acyl-CoA synthetases (fatty acid activation proteins, Faaps), Faa1p and Faa4p activate exogenously derived fatty acids. Acyl-CoA metabolism plays a critical role in regulating protein N-myristoylation by the essential enzyme, myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). In this report, we have examined whether Faa1p and Faa4p have distinct roles in affecting protein N-myristoylation as cells transition from growth in rich media to a growth-arrested state during nutrient deprivation (stationary phase). The colony-forming potential of 10 isogenic strains was defined as a function of time spent in stationary phase. These strains contained either a wild type or mutant NMTI allele, and wild type or null alleles of each FAA. Only the combination of the Nmt mutant (nmt451Dp; reduced affinity for myristoyl-CoA) and loss of Faa4p produced a dramatic loss of colony-forming units (CFU). The progressive millionfold reduction in CFU was associated with a deficiency in protein N-myristoylation that first appeared during log arithmic growth, worsened through the post-diauxic phase, and became extreme in stationary phase. Northern and Western blot analyses plus N-myristoyltransferase assays showed that Nmt is normally present only during the log and diauxic/post-diauxic periods, indicating that N-myristoylproteins present in stationary phase are "inherited" from these earlier phases. Moreover, FAA4 is the only FAA induced during the critical diauxic/early post-diauxic transition. Although substitution of nmt1-451D for NMT1 results in deficiencies in protein N-myristoylation, these deficiencies are modest and limited by compensatory responses that include augmented expression of nmt1-451D and precocious induction of FAA4 in log phase. Loss of Faa4p from nmt1-451D cells severely compromises their capacity to adequately myristoylate Nmt substrates prior to entry into stationary phase since none of the other Faaps are able to functionally compensate for its absence. To identify Nmt1p substrates that may affect maintenance of proliferative potential during stationary phase, we searched the yeast genome for known and putative N-myristoylproteins. Of the 64 genes found, 48 were successfully deleted in NMT1 cells. Removal of any one of the following nine substrates produced a loss of CFU similar to that observed in nmt1-451Dfaa4 Delta cells: Arf1p, Arf2p, Sip2p, Van1p, Ptc2p, YBL049W (homology to Snf7p), YJR114W, YKR007W, and YMR077C. These proteins provide opportunities to further define the molecular mechanisms that regulate survival during stationary phase.