Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells

The formation of intra- and interchain disulphide bonds constitutes an inte gral part of the maturation of most secretory and membrane-bound proteins i n the endoplasmic reticulum(1,2). Evidence indicates that members of the pr otein disulphide isomerase (PDI) superfamily are part of the machinery need ed for proper oxidation and isomerization of disulphide bonds(3-6). Models based on in vitro studies predict that the formation of mixed disulphide bo nds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide(7). W hether this is how thiol oxidoreductases work inside the endoplasmic reticu lum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate pro teins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding(7,8). Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidored uctases PDI and ERp57 are directly involved in disulphide oxidation and iso merization, and, together with the lectins calnexin and calreticulin, are c entral in glycoprotein folding in the endoplasmic reticulum of mammalian ce lls.