ER60/ERp57 forms disulfide-bonded intermediates with MHC class I heavy chain.

Abstract

SPECIFIC AIMSIn this study, we investigated the role of ER60 (ERp57/GRP58) in the formation of disulfide bonds within the major histocompatibility (MHC) class I heavy chain. We also examined the role of calnexin in recruiting ER60 into early folding complexes with the nascent class I heavy chain (HC).PRINCIPAL FINDINGS1. ER60/ERp57 forms disulfide-bonded intermediates with class I heavy chainMouse EL4 cells were metabolically labeled for 15 min with [35S] cysteine/methionine. After labeling, the cells were washed in PBS containing N-ethyl-maleimide (NEM), a membrane-permeable alkylating agent, and lysed in 1% digitonin lysis buffer containing NEM to trap unreacted cysteines. Protein complexes containing ER60 were isolated by immunoprecipitation using anti-ER60 sera. To identify a disulfide-bonded intermediate, the complexes were resolved on a 2-dimensional gel system using nonreducing gels in the first dimension. Proteins were resolved on 8% polyacrylamide gels in capillary tubes. The gels were then extru...