Protease B of Saccharomyces cerevisiae: isolation and regulation of the PRB1 structural gene.
Protease B of Saccharomyces cerevisiae: isolation and regulation of the PRB1 structural gene.
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DOI：
10.1093/genetics/115.2.255
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发表时间：
1987-02
期刊：
影响因子：
3.3
通讯作者：
C. Moehle;M. W. Aynardi;M. Kolodny;F. J. Park;E. W. Jones
中科院分区：
文献类型：
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作者：
C. Moehle;M. W. Aynardi;M. Kolodny;F. J. Park;E. W. Jones
我们通过 prb1-1122 突变的互补，从基因组文库中分离出了酿酒酵母液泡蛋白酶 B 的结构基因 PRB1。缺失分析将互补活性定位于 3.2 KB 对 XhoI-HindIII 限制酶片段。该片段用于鉴定 2.3 KB 的 mRNA。 S1核酸内切酶图谱表明mRNA和基因是共线的。没有检测到内含子。 mRNA的大小足以编码约69,000分子量的蛋白质，这个数字远大于成熟酶（相当于30,000蛋白质分子量）或唯一报道的前体（相当于39,000蛋白质分子量）。这些结果表明，将初始糖基化翻译产物转化为成熟的蛋白酶 B 可能需要超出被认为由蛋白酶 A 催化的蛋白水解加工步骤。仅当细胞耗尽葡萄糖供应并进入双峰平台时，PRB1 mRNA 才会大量产生。 PRB1 转录和蛋白酶 B 活性表达之间存在较长的时间滞后。删除约83%编码区的缺失被构建为二倍体杂合子。带有缺失的孢子会发芽，以正常速率生长成菌落，并且除了蛋白酶 B 缺陷之外没有明显的表型。
We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation. Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment. The fragment was used to identify a 2.3-kilobase mRNA. S1 endonuclease mapping indicated that the mRNA and the gene were colinear. No introns were detected. The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (congruent to 30,000 protein molecular weight) or the sole reported precursor (congruent to 39,000 protein molecular weight). These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B. The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau. There is an extended time lag between PRB1 transcription and expression of protease B activity. A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote. Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.