Jamie-Lee Berry, Neil J. Bulleid

This work explores the role of the thiol-oxidoreductase ERp57 in the post-translational oxidative folding of the low-density lipoprotein receptor (LDL-R), a cell-surface glycoprotein responsible for the uptake of cholesterol from plasma. The LDL-R provides a general model to analyse oxidative folding of multi-domain proteins in the endoplasmic reticulum; yet its folding pathway is also of specific interest as a high proportion of mutations in disulphide-rich domains of the protein are evident in familial hypercholesterolemia. Previous studies have suggested that the LDL-R forms a set of distinct non-native disulphide intermediates during folding, which are extensively isomerized prior to secretion of the native conformer. In addition, ERp57 has been suggested to be predominantly reduced in vivo and to form a mixed disulphide with the LDL-R. In this study, the LDL-R was expressed in both wild-type cells and those lacking the thiol-oxidoreductase ERp57 under conditions that prevent disulphide formation. The protein was then allowed to fold under oxidizing conditions, and samples taken at various timepoints. The electrophoretic mobility of folding intermediates from knock-out cells was compared with that of wild-type cells. The results show that dissimilar disulphide intermediates form between the two cell types, particularly during early stages of folding. A mutant form of ERp57, able to form but unable to resolve mixed disulphides, was also found to form mixed disulphides with the LDL-R. The results signify the requirement for ERp57 in oxidative folding of the LDL-R and also suggest that non-native disulphide intermediates may be central to the process of multi-domain protein folding.