Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003.
Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003.
J Biol Chem. 2019 Dec 13;294(50):18992-19011. doi: 10.1074/jbc.RA119.010450. Epub 2019 Oct 29.
The protein quality control machinery of the endoplasmic reticulum (ERQC) ensures that client proteins are properly folded. ERQC substrates may be recognized as nonnative by the presence of exposed hydrophobic surfaces, free thiols, or processed -glycans. How these features dictate which ERQC pathways engage a given substrate is poorly understood. Here, using metabolic labeling, immunoprecipitations, various biochemical assays, and the human serpin antithrombin III (ATIII) as a model, we explored the role of ERQC systems in mammalian cells. Although ATIII has -glycans and a hydrophobic core, we found that its quality control depended solely on free thiol content. Mutagenesis of all six Cys residues in ATIII to Ala resulted in its efficient secretion even though the product was not natively folded. ATIII variants with free thiols were retained in the endoplasmic reticulum but not degraded. These results provide insight into the hierarchy of ERQC systems and reveal a fundamental vulnerability of ERQC in a case of reliance on the thiol-dependent quality control pathway.
内质网（ER）的蛋白质质量控制机制可确保客户蛋白正确折叠。ERQC 底物可能通过暴露的疏水面、游离巯基或加工的糖基化来被识别为非天然的。这些特征如何决定 ERQC 途径与特定底物结合，目前还知之甚少。在这里，我们使用代谢标记、免疫沉淀、各种生化测定以及人丝氨酸蛋白酶抑制剂抗凝血酶 III（ATIII）作为模型，探索了 ERQC 系统在哺乳动物细胞中的作用。尽管 ATIII 具有糖基化和疏水性核心，但我们发现其质量控制仅取决于游离巯基含量。将 ATIII 中的六个半胱氨酸残基突变为丙氨酸，即使产物没有天然折叠，也能使其有效地分泌。具有游离巯基的 ATIII 变体在 ER 中被保留但不会降解。这些结果提供了 ERQC 系统的层次结构的深入了解，并揭示了在依赖巯基依赖的质量控制途径的情况下 ERQC 的基本脆弱性。
