Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum.
Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum.
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DOI：
10.1083/jcb.138.6.1229
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发表时间：
1997-09-22
期刊：
影响因子：
--
通讯作者：
Winther JR
中科院分区：
文献类型：
--
作者：
Holst B;Tachibana C;Winther JR
蛋白质二硫键异构酶（PDI）的功能方面已经在酵母体内进行了研究。PDI含有两个硫氧还蛋白样结构域a和a′，每个结构域都含有一个活性位点CXXC基序。这两个结构域的相对重要性通过使每个结构域突变为SGAS而失活来分析。这些突变对生长没有显著影响。然而，结构域并不等同，因为羧肽酶Y（CPY）在体内的折叠速率会因a结构域而非a′结构域的失活而降低。为了研究PDI氧化还原电位的相关性，对每个CGHC活性位点的G和H位置进行随机诱变。根据其在含有浓度不断增加的DTT的培养基上的生长表型对所得突变体PDI进行排名。突变体中的CPY折叠速率与DTT敏感性显示出相同的排名，表明PDI的氧化能力是体内折叠的重要因素。具有不能自行进行氧化反应的PDI的突变体（CGHS）具有强烈降低的生长速率。然而，生长速率与CPY折叠不相关，表明最佳生长所需的蛋白质依赖于氧化的PDI。过表达酵母PDI同源物EUG 1的pdi 1缺失菌株是活的。将野生型Eug 1 p C（L/I）HS活性位点序列交换为C（L/I）HC显著增加了生长速率，然而，进一步突出了氧化功能对最佳生长的重要性。
Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.