Nozawa Michiyo, Takahashi Toshinari, Hara Shodo, Mizoguchi Haruhiko
General Research Laboratory of Kiku-Masamune Sake Brewing Co. Ltd., 1-8-6 Uozaki-nishimachi, Higashinada-ku, Kobe 658, Japan.
J Biosci Bioeng. 2002;93(3):288-95. doi: 10.1263/jbb.93.288.
It is well known that sake yeast has a high tolerance for ethanol, as compared to baker's yeast. To investigate the relationship between the ethanol tolerance of sake yeast and the palmitoyl-CoA pool for protein modification, the growth of yeast cells with depletion of the palmitoyl-CoA pool was monitored in the presence of ethanol. The overexpression of SNC1 was used to achieve the depletion of the palmitoyl-CoA pool, because the loss of Snc palmitoylation does not affect the general growth characteristics of yeast and does not interfere with the secretory processes (Couve, A. et al., Proc. Natl. Acad. Sci. USA, 92, 5987-5991 (1995)). Although the sake yeast UT-1 exhibited much better growth in the presence of ethanol than laboratory strains, the overexpression of Snc1 was accompanied by sparse growth with increasing ethanol concentration. Exogenous palmitic acid rescued the poor growth caused by Snc1 overexpression, and the overexpression of Snc1(ser95) (which could not palmitoylated) had no effect on the growth characteristics of strain UT-1, suggesting that the poor growth with Snc1 overexpression was due to an overall increase in proteins in the unpalmitoylated form. To ascertain that fatty acid activation has a distinct role in the growth of yeast in the presence of ethanol, FAA genes encoding long chain acyl-CoA synthetases were overexpressed in combination with snc1 overexpression. Interestingly, the growth defect caused by snc1 overexpression was rescued by the overexpression of FAA4, but not of FAA1, which plays a predominant role in laboratory strains. On the contrary, disruption of faa1 led to faster growth in the presence of ethanol. These results suggest that Faa1p and Faa4p play reciprocal roles in regulating protein modification during growth in the presence of ethanol, since Faa1p and Faa4p both function to incorporate palmitic acid into phospholipids and neutral lipids. Moreover, Northern hybridization analysis revealed that faa1 mRNA was expressed strongly in a laboratory strain, and weakly in the sake yeast strain K-7 which exhibited good growth in the presence of ethanol. The combination of the disruption of faa1 and exogenously supplied palmitic acid was highly effective for growth in the presence of ethanol even under the normal snc1 expression level, implying that activation of exogenous palmitic acid by Faa4p is of particular importance in growth in ethanol.
众所周知，与面包酵母相比，清酒酵母对乙醇具有较高的耐受性。为了研究清酒酵母的乙醇耐受性与用于蛋白质修饰的棕榈酰辅酶A库之间的关系，在乙醇存在的情况下监测了棕榈酰辅酶A库耗尽的酵母细胞的生长情况。使用SNC1的过表达来实现棕榈酰辅酶A库的耗尽，因为Snc棕榈酰化的缺失不会影响酵母的一般生长特性，也不会干扰分泌过程（库夫，A.等人，《美国国家科学院院刊》，92，5987 - 5991（1995））。尽管清酒酵母UT - 1在乙醇存在下的生长比实验室菌株好得多，但随着乙醇浓度的增加，Snc1的过表达伴随着稀疏生长。外源性棕榈酸挽救了由Snc1过表达引起的生长不良，并且Snc1(ser95)（不能进行棕榈酰化）的过表达对UT - 1菌株的生长特性没有影响，这表明Snc1过表达导致的生长不良是由于未棕榈酰化形式的蛋白质总体增加。为了确定脂肪酸活化在乙醇存在下酵母生长中具有独特作用，将编码长链酰基辅酶A合成酶的FAA基因与snc1过表达联合过表达。有趣的是，snc1过表达引起的生长缺陷通过FAA4的过表达得到挽救，但FAA1的过表达则不能，FAA1在实验室菌株中起主要作用。相反，faa1的破坏导致在乙醇存在下生长更快。这些结果表明，Faa1p和Faa4p在乙醇存在下的生长过程中在调节蛋白质修饰方面发挥相反的作用，因为Faa1p和Faa4p都起到将棕榈酸掺入磷脂和中性脂质中的作用。此外，Northern杂交分析表明，faa1 mRNA在实验室菌株中强烈表达，而在清酒酵母菌株K - 7中微弱表达，K - 7在乙醇存在下表现出良好的生长。即使在正常的snc1表达水平下，faa1的破坏与外源性提供的棕榈酸的组合对于在乙醇存在下的生长也非常有效，这意味着Faa4p对外源性棕榈酸的活化在乙醇中的生长中特别重要。
