Purification and Characterization of the Human KDEL Receptor

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• 作者：Andreas A. Scheel and Hugh R. B. Pelham
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：August 6, 1996
• 年：1996
• 卷：35
• 期：31
• 页码：10203 - 10209
• 全文大小：396K
• 年卷期：v.35,no.31(August 6, 1996)
• ISSN：1520-4995

文摘

Retention of soluble endoplasmic reticulum (ER) proteins isensured by their continuous retrievalfrom subsequent compartments in the secretory pathway. Soluble ERproteins which escape to the Golgiapparatus bind to the KDEL receptor, a seven-transmembrane receptor,and are then returned to theendoplasmic reticulum. We have overexpressed the human KDELreceptor in insect cells using thebaculovirus system. Infected cells accumulate large amounts offunctional receptor as judged by a ligandbinding assay. A hexahistidine-tagged version of the receptorcould be purified in a single step to nearhomogeneity with high yield. After reconstitution of purified KDELreceptor into liposomes, a similaraffinity and pH dependence for the binding of KDEL peptides wasobserved compared to the receptor inits natural environment, indicating that purified KDEL receptor issufficient for specific and pH-sensitivebinding of KDEL ligands. Determination of the receptor affinity indifferent lipid environments revealedthat the receptor affinity is only slightly influenced by its lipidenvironment, suggesting that regulation ofthe receptor affinity by its surrounding lipids does not play a crucialrole for the sorting of KDEL proteins.