Zhang J X, Braakman I, Matlack K E, Helenius A
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510-8002, USA.
Mol Biol Cell. 1997 Oct;8(10):1943-54. doi: 10.1091/mbc.8.10.1943.
Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, < 2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intra-chain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.
与正确折叠和组装的蛋白质不同，大多数错误折叠和组装不完全的蛋白质会保留在哺乳动物细胞的内质网中，并在不转运至高尔基体复合体的情况下被降解。为了分析这种独特分选过程及其保真度背后的机制，我们确定了流感血凝素C末端截短片段的命运。通过向感染流感病毒的组织培养细胞中添加低浓度嘌呤霉素，产生了一系列不同的片段。在产生的片段中，不到2%被分泌，这表明用于检测新合成蛋白质缺陷的系统相当严格。大多数分泌的片段对应于病毒刺突糖蛋白内的折叠结构域。保留的片段获得了具有链内二硫键和构象依赖性抗原表位的部分折叠结构。它们与两种凝集素样内质网伴侣蛋白（钙连蛋白和钙网蛋白）结合，但不与BiP/GRP78结合。通过添加栗精胺抑制与钙连蛋白和钙网蛋白的结合，显著增加了片段的分泌。然而，这也导致了与BiP/GRP78的结合。这些结果表明，与钙连蛋白和钙网蛋白的结合参与了片段的保留。它们还表明，BiP/GRP78可以作为钙连蛋白和钙网蛋白在保留片段方面的后备蛋白。总之，结果表明分泌途径中的质量控制系统对折叠缺陷有效且敏感，并且它涉及与内质网伴侣蛋白的多种相互作用。
