Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae
Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae
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DOI：
10.1091/mbc.8.11.2267
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发表时间：
1997-11-01
影响因子：
3.3
通讯作者：
Heitman, J
中科院分区：
文献类型：
--
作者：
Dolinski, K;Scholz, C;Heitman, J
亲环素和FK 506结合蛋白（FKBP）加速顺-反肽基脯氨酰异构化，并结合并介导免疫抑制剂环孢菌素A和FK 506的作用。然而，这些蛋白质的正常细胞功能是未知的。我们改变了活性位点的FKBP 12和线粒体亲环素从酵母酿酒酵母通过引入突变先前报道的，以抑制这些酶。令人惊讶的是，这些突变酶中的大多数在体内具有生物活性。与以前的报道雅阁，在标准肽底物-胰凝乳蛋白酶偶联的体外测定中，所有的突变酶都具有很少或没有可检测的脯氨酰异构酶活性。然而，在省略蛋白酶的该测定的变型中，突变体酶表现出显著水平的脯氨酰异构酶活性（野生型的5-20%），揭示了这些突变赋予对蛋白酶消化的敏感性，并且脯氨酰异构酶活性的经典体外测定可能是误导性的。此外，突变酶表现出接近野生型的活性与两种蛋白质底物，二氢叶酸还原酶和核糖核酸酶T1，其折叠是加速脯氨酰异构酶。因此，以前鉴定的许多亲环蛋白和FKBP 12“活性位点”突变体在很大程度上是活性的，但对蛋白酶敏感，这与我们的发现雅阁，即这些突变体在体内显示野生型功能。一种线粒体亲环蛋白突变体（R73 A）以及野生型人FKBP 12酶在体外催化蛋白质折叠，但在酵母中缺乏体内生物活性。我们的研究结果提供的证据表明，脯氨酰异构酶的活性和其他结构特征与FKBP和亲环素在体内的功能，并建议谨慎使用这些活性位点突变研究FKBP和亲环素的功能。
Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 ''active-site'' mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.