Two conserved cysteine triads in human Ero1α cooperate for efficient disulfide bond formation in the endoplasmic reticulum
Two conserved cysteine triads in human Ero1α cooperate for efficient disulfide bond formation in the endoplasmic reticulum
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DOI：
10.1074/jbc.m403192200
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发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Sitia, R
中科院分区：
文献类型：
--
作者：
Bertoli, G;Simmen, T;Sitia, R
人Ero 1 α是一种内质网（ER）驻留蛋白，负责蛋白质二硫键异构酶（PDI）氧化。为了阐明其功能的分子机制，我们产生了一组半胱氨酸置换突变体，并分析了它们的能力：1）补充温度敏感的酵母Ero 1突变体，2）有利于哺乳动物细胞中的氧化折叠，3）与PDI和ERp 44形成混合二硫化物，以及4）采用特征性氧化还原依赖性构象。我们的研究结果表明，两个基本的半胱氨酸三联体（Cys（85）-Cys（94）-Cys（99）和Cys（391）-Cys（394）-Cys（397））在电子转移中合作，Cys（94）可能与PDI形成混合二硫化物。当三联体中的关键残基突变时，出现显性阴性表型（Cys（394）、Cys（397）和较小程度的Cys（99））。替换任一三联体（Cys（85）或Cys（391））中的第一个半胱氨酸产生具有较弱活性的突变体。此外，突变Cys（85）或Cys（391），但不突变Cys（397），可逆转C394 A突变体的显性阴性表型。这些发现表明，依赖于Cys（85）和Cys（391）的两个三联体之间的相互作用对Ero 1 alpha功能很重要，可能稳定了有效PDI氧化的平台。
Human Ero1alpha is an endoplasmic reticulum (ER)-resident protein responsible for protein disulfide isomerase (PDI) oxidation. To clarify the molecular mechanisms underlying its function, we generated a panel of cysteine replacement mutants and analyzed their capability of: 1) complementing a temperature-sensitive yeast Ero1 mutant, 2) favoring oxidative folding in mammalian cells, 3) forming mixed disulfides with PDI and ERp44, and 4) adopting characteristic redox-dependent conformations. Our results reveal that two essential cysteine triads (Cys(85)-Cys(94)-Cys(99) and Cys(391)-Cys(394)-Cys(397)) cooperate in electron transfer, with Cys(94) likely forming mixed disulfides with PDI. Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys(394), Cys(397), and to a lesser extent Cys(99)). Replacing the first cysteine in either triad (Cys(85) or Cys(391)) generates mutants with weaker activity. In addition, mutating either Cys(85) or Cys(391), but not Cys(397), reverts the dominant negative phenotype of the C394A mutant. These findings suggest that interactions between the two triads, dependent on Cys(85) and Cys(391), are important for Ero1alpha function, possibly stabilizing a platform for efficient PDI oxidation.