Kopp M, Müller H, Holzer H
Biochemisches Institut, Universität Freiburg, Germany.
J Biol Chem. 1993 Mar 5;268(7):4766-74.
Neutral trehalase (EC 3.2.1.28) is a trehalose hydrolyzing enzyme of the yeast Saccharomyces cerevisiae (App, H., and Holzer, H. (1989) J. Biol. Chem. 264, 17583-17588). The gene of neutral trehalase was cloned by complementation of a neutral trehalase-deficient yeast mutant which was obtained by ethylmethanesulfonate mutagenesis. Three mutants without detectable neutral trehalase activity were obtained and characterized by tetrad analysis and found to belong to the same complementation group. The mutants were transformed with a S. cerevisiae genomic library in YEp24. Two overlapping plasmids were isolated, containing the neutral trehalase gene NTH1 with an open reading frame of 2079 base pairs (bp), encoding a protein of 693 amino acids, corresponding to a molecular mass of 79,569 Da. Several putative TATA boxes were found in the 5'-nontranslated region of the NTH1 gene. In positions -652 to -641 a possible binding sequence for the MIG1 protein, a multicopy inhibitor of the GAL1 promotor, which also binds to the promotor sequences of the SUC2 and the FBP1 gene, was found. The start codon of the neutral trehalase is located about 2500 bp upstream of the centromere 4 consensus sequence elements I, II, and III (Mann, C., and Davis, R. W. (1986) Mol. Cell. Biol. 6, 241-245). Vicinity to a centromere is known to have a depressing influence on the number of plasmid copies per cell. This probably explains why transformation with pNTH does not lead to overexpression of neutral trehalase. The four consensus sequences AATAAA contained in the centromeric elements and reconfirmed by our sequencing data might be polyadenylation signals for NTH1-mRNA transcription termination. Northern blot analysis yielded a single mRNA species of approximately 2.3 kilobase(s). The neutral trehalase protein has a putative cAMP-dependent phosphorylation consensus sequence RRGS from amino acid positions 22-25. Therefore, the previously described activation of neutral trehalase by cAMP-dependent phosphorylation is probably due to phosphorylation of serine 25. Three potential N-glycosylation sites (Asn-X-Ser/Thr) occur in the open reading frame of the neutral trehalase gene. However, no evidence for glycosylation could be detected by Western blotting.(ABSTRACT TRUNCATED AT 400 WORDS)
中性海藻糖酶（EC 3.2.1.28）是酿酒酵母中的一种海藻糖水解酶（App, H., and Holzer, H. (1989) J. Biol. Chem. 264, 17583 - 17588）。中性海藻糖酶基因是通过对一个中性海藻糖酶缺陷型酵母突变体进行互补克隆得到的，该突变体是通过甲磺酸乙酯诱变获得的。获得了三个没有可检测到的中性海藻糖酶活性的突变体，并通过四分体分析进行了表征，发现它们属于同一互补群。用YEp24中的酿酒酵母基因组文库转化这些突变体。分离出两个重叠质粒，其中包含中性海藻糖酶基因NTH1，其开放阅读框为2079个碱基对（bp），编码一个693个氨基酸的蛋白质，分子量为79,569 Da。在NTH1基因的5'非翻译区发现了几个假定的TATA盒。在 - 652至 - 641位置发现了一个可能的MIG1蛋白结合序列，MIG1蛋白是GAL1启动子的多拷贝抑制剂，它也与SUC2和FBP1基因的启动子序列结合。中性海藻糖酶的起始密码子位于着丝粒4共有序列元件I、II和III（Mann, C., and Davis, R. W. (1986) Mol. Cell. Biol. 6, 241 - 245）上游约2500 bp处。已知靠近着丝粒会对每个细胞中的质粒拷贝数产生抑制作用。这可能解释了为什么用pNTH转化不会导致中性海藻糖酶的过表达。着丝粒元件中包含的四个共有序列AATAAA并经我们的测序数据再次确认，可能是NTH1 - mRNA转录终止的聚腺苷酸化信号。Northern印迹分析产生了一种约2.3千碱基的单一mRNA种类。中性海藻糖酶蛋白在氨基酸位置22 - 25处有一个假定的cAMP依赖性磷酸化共有序列RRGS。因此，先前描述的cAMP依赖性磷酸化对中性海藻糖酶的激活可能是由于丝氨酸25的磷酸化。在中性海藻糖酶基因的开放阅读框中出现了三个潜在的N - 糖基化位点（Asn - X - Ser/Thr）。然而，通过蛋白质免疫印迹法未检测到糖基化的证据。（摘要截断于400字）
