Luz J M, Lennarz W J
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215, USA.
Biochem Biophys Res Commun. 1998 Jul 30;248(3):621-7. doi: 10.1006/bbrc.1998.8992.
Protein disulfide isomerase (PDI), the product of the essential PDI1 gene of Saccharomyces cerevisiae catalyzes oxidization of thiols, reduction of disulfide bonds, and isomerization of disulfides. It can also act as a chaperone to facilitate folding of denatured proteins. The protein has 6 cysteine (Cys) residues. Four of these Cys are part of the 2 thioredoxin-like catalytic sites (-CGHC-), one of which is located near the N- and the other near the C-terminus. In addition, it has 2 non-active site Cys near the N-terminus. The function of these non-active site Cys of yeast PDI is poorly understood. Whereas in yeast PDI, these Cys residues are in the vicinity of the N-terminal-most active site, in mammalian PDI their position is closer to the C-terminal-most active site. We have examined their role and that of the active site cysteines by constructing an extensive set of mutants in which the Cys were systematically replaced by Ser. As reported earlier, the N-terminal Cys of the two active sites sequences of yeast PDI were found to be required for cell viability, but mutation of the C-terminal Cys to Ser in the two active sites was not lethal. We found that replacement of the two non-active site Cys with Ser did not affect cell viability, but in the case of the double mutant in which both Cys were replaced by Ser the processing and secretion of CPY was impaired.
蛋白质二硫键异构酶（PDI）是酿酒酵母必需基因PDI1的产物，可催化硫醇的氧化、二硫键的还原以及二硫键的异构化。它还可作为伴侣蛋白促进变性蛋白质的折叠。该蛋白有6个半胱氨酸（Cys）残基。其中4个Cys是2个硫氧还蛋白样催化位点（-CGHC-）的一部分，其中一个位于N端附近，另一个位于C端附近。此外，它在N端附近还有2个非活性位点Cys。对酵母PDI这些非活性位点Cys的功能了解甚少。在酵母PDI中，这些Cys残基位于最靠近N端的活性位点附近，而在哺乳动物PDI中，它们的位置更靠近最靠近C端的活性位点。我们通过构建一系列广泛的突变体对它们以及活性位点半胱氨酸的作用进行了研究，在这些突变体中，Cys被系统地替换为Ser。如先前报道，酵母PDI两个活性位点序列的N端Cys被发现是细胞存活所必需的，但两个活性位点中C端Cys突变为Ser并不致命。我们发现将两个非活性位点Cys替换为Ser不影响细胞活力，但在两个Cys都被替换为Ser的双突变体中，羧肽酶Y（CPY）的加工和分泌受损。
