Scheel A A, Pelham H R
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Biochemistry. 1996 Aug 6;35(31):10203-9. doi: 10.1021/bi960807x.
Retention of soluble endoplasmic reticulum (ER) proteins is ensured by their continuous retrieval from subsequent compartments in the secretory pathway. Soluble ER proteins which escape to the Golgi apparatus bind to the KDEL receptor, a seven-transmembrane receptor, and are then returned to the endoplasmic reticulum. We have overexpressed the human KDEL receptor in insect cells using the baculovirus system. Infected cells accumulate large amounts of functional receptor as judged by a ligand binding assay. A hexahistidine-tagged version of the receptor could be purified in a single step to near homogeneity with high yield. After reconstitution of purified KDEL receptor into liposomes, a similar affinity and pH dependence for the binding of KDEL peptides was observed compared to the receptor in its natural environment, indicating that purified KDEL receptor is sufficient for specific and pH-sensitive binding of KDEL ligands. Determination of the receptor affinity in different lipid environments revealed that the receptor affinity is only slightly influenced by its lipid environment, suggesting that regulation of the receptor affinity by its surrounding lipids does not play a crucial role for the sorting of KDEL proteins.
通过从分泌途径的后续区室持续回收，可确保可溶性内质网（ER）蛋白的保留。逃逸到高尔基体的可溶性ER蛋白与KDEL受体（一种七跨膜受体）结合，然后返回内质网。我们使用杆状病毒系统在昆虫细胞中过表达了人KDEL受体。通过配体结合试验判断，感染的细胞积累了大量功能性受体。带有六组氨酸标签的受体版本可以一步纯化至高纯度且高产率。将纯化的KDEL受体重建到脂质体中后，与天然环境中的受体相比，观察到其对KDEL肽结合具有相似的亲和力和pH依赖性，表明纯化的KDEL受体足以实现KDEL配体的特异性和pH敏感结合。在不同脂质环境中测定受体亲和力表明，受体亲和力仅受其脂质环境的轻微影响，这表明其周围脂质对受体亲和力的调节对KDEL蛋白的分选不起关键作用。
