REGULATORY SUBUNIT (CNB1 GENE-PRODUCT) OF YEAST CA2+/CALMODULIN-DEPENDENT PHOSPHOPROTEIN PHOSPHATASES IS REQUIRED FOR ADAPTATION TO PHEROMONE
REGULATORY SUBUNIT (CNB1 GENE-PRODUCT) OF YEAST CA2+/CALMODULIN-DEPENDENT PHOSPHOPROTEIN PHOSPHATASES IS REQUIRED FOR ADAPTATION TO PHEROMONE
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DOI：
10.1128/mcb.12.8.3460
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发表时间：
1992-08-01
影响因子：
5.3
通讯作者：
THORNER, J
中科院分区：
文献类型：
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作者：
CYERT, MS;THORNER, J
通过使用一种检测钙调神经磷酸酶（一种Ca2+/钙调蛋白依赖的磷酸蛋白磷酸酶）的检测方法，这种酶从酵母的提取物中纯化了大约5000倍。Cna1p和Cna2p是编码钙调磷酸酶催化(A)亚基的两个酵母基因的产物，是纯化组分的主要成分。表观分子质量为16 kDa的第三个突出成分显示出一些特性，包括能够结合Ca-45(2+)，这是哺乳动物钙调神经磷酸酶的调节(B)亚基的特征，并被抗牛钙调神经磷酸酶的抗血清识别。这些抗体用于从载体lambda-gt11的酵母表达文库中分离编码该蛋白的结构基因CNB1。CNB1的核苷酸序列预测了一个与哺乳动物钙调磷酸酶B亚基长度相似且氨基酸序列高度相关（56%同源性）的多肽。与高等细胞中的对应物一样，酵母的Cnb1p在其N端发生肉豆蔻酰化。缺乏Cnb1p或所有三种钙调磷酸酶亚基（Cna1p， Cna2p和Cnb1p）的突变体是可存活的。尽管Cna1p和Cna2p在正常水平，但cnb1-DELTA突变体的提取物中没有检测到钙调磷酸酶活性，这表明B亚基是体外酶活性充分发挥所必需的。正如之前在MATa cna1 cna2双突变体中观察到的那样，MATa cnb1突变体从α因子诱导的生长停滞中恢复的能力存在缺陷。因此，钙调磷酸酶在体内促进单倍体酵母细胞对信息素的适应也需要B亚基。
By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind Ca-45(2+), that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda-gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1-DELTA mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.