Hiser L, Basson M E, Rine J
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Biol Chem. 1994 Dec 16;269(50):31383-9.
Two recessive alleles of ERG10 and three temperature-sensitive recessive alleles of HMG1 (3-hydroxy-3-methyl-glutaryl-CoA reductase isoenzyme 1) were isolated in a screen for mevalonate auxotrophs in Saccharomyces cerevisiae. The essential, single-copy ERG10 gene was cloned by complementation of the temperature-sensitive phenotype of erg10-21. The 1,194-base pair continuous open reading frame, encoding a 398-amino acid polypeptide with a calculated molecular mass of 41,681 daltons, was demonstrated to encode cytoplasmic aceto-acetyl-CoA thiolase. Acetoacetyl-CoA thiolase activity corresponded to the number of copies of ERG10 present in cell extracts, and null alleles of ERG10 produced no detectable acetoacetyl-CoA thiolase enzyme activity. The deduced amino acid sequence was 40-95% identical to acetoacetyl-CoA thiolases from other organisms. This identity included the active site cysteines located at amino acids 91 and 384 in the Erg10 protein.
在酿酒酵母中筛选甲羟戊酸营养缺陷型时，分离出了ERG10的两个隐性等位基因和HMG1（3-羟基-3-甲基戊二酰辅酶A还原酶同工酶1）的三个温度敏感型隐性等位基因。通过对erg10-21温度敏感表型的互补作用，克隆了必需的单拷贝ERG10基因。1194个碱基对的连续开放阅读框编码一个398个氨基酸的多肽，计算分子量为41681道尔顿，已证明其编码细胞质乙酰乙酰辅酶A硫解酶。乙酰乙酰辅酶A硫解酶活性与细胞提取物中存在的ERG10拷贝数相对应，ERG10的无效等位基因未产生可检测到的乙酰乙酰辅酶A硫解酶活性。推导的氨基酸序列与其他生物的乙酰乙酰辅酶A硫解酶有40%-95%的同一性。这种同一性包括位于Erg10蛋白第91和384位氨基酸处的活性位点半胱氨酸。
