trends in CELL BIOLOGY (Vol. 10) May 2000 0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. 203 PII: S0962-8924(00)01745-1 The formation of disulphide bonds between the cor- rect pairs of cysteine residues is essential for the fold- ing and stability of many proteins that are secreted or localized to organelles of the secretory pathway. Nearly 40 years ago, the seminal work of Anfinsen and colleagues on the in vitro refolding of reduced, denatured ribonuclease A demonstrated that native disulphide bond formation can occur spontaneously 1 . The early observation that disulphide bond for- mation proceeded much more slowly in air than in living cells implied the existence of catalysts for oxidative protein folding. The minimal requirements for efficient oxida- tive refolding in vitro have since been defined as a redox buffer containing both oxidizing and reduc- ing equivalents as well as an enzymatic catalyst for thiol–disulphide exchange. Standard assays for oxidative refolding employ glutathione redox buffers in which oxidized glutathione (GSSG) provides the oxidizing equivalents necessary for protein disul- phide bond formation. Under these conditions, the redox potential of the assay buffer, defined by the ratio [GSH] 2 /[GSSG], determines the overall rate of oxidative refolding 2,3 . The search for enzymatic catalysts of oxidative refolding led to the isolation of protein disulphide isomerase (PDI) 4 . Intensive investigation of the activities of PDI in vitro has since shown that the enzyme can catalyse the formation, reduction or isomerization of disulphide bonds depending upon the redox conditions of the assay and the nature of the substrate protein 5 . The activity of PDI depends on two pairs of cysteines, each of which are found in the motif Cys-x a -x b -Cys within a domain homolo- gous to thioredoxin 6 . When the active-site cysteines of PDI are present in disulphide (oxidized) form, the enzyme can transfer disulphide bonds directly to substrate proteins, suggesting a physiological role for PDI as a protein dithiol oxidase 7 . However, when the active-site cysteines of PDI are present in dithiol (reduced) form, the enzyme is suited for catalysis of disulphide reshuffling. PDI is reduced in those redox buffers adjusted to give optimal refolding rates in vitro, and this correlation has drawn attention to the isomerase activity of the enzyme 8 . A role for PDI in the catalysis of native disulphide bond formation in the endoplasmic reticulum (ER) was first established by mutational analysis in yeast, where the PDI1 gene was shown to be essential for cell viability and for oxidative protein folding 9,10 . In eukaryotic cells, protein disulphide bond for- mation proceeds within the lumen of the ER, where protein oxidation initiates upon the translocation of nascent peptide chains into the ER lumen 11 . The redox state of the ER is more oxidizing than that of the cytosol, a difference that favours the formation of structural protein disulphide bonds, and that is re- flected in the relatively high intralumenal concen- tration of GSSG 12,13 . A net influx of oxidizing equiva- lents into the ER lumen is needed to support the rapid transit of secretory proteins through the ER, and to maintain a high concentration of GSSG within this organelle 12 . However, until very recently, the source of oxidizing equivalents utilized for disulphide bond formation in the ER was unclear. Genetic analy- sis in Saccharomyces cerevisiae has now defined the core pathway for protein disulphide bond formation in the eukaryotic ER. A pathway for protein disulphide bond formation in the ER A genetic dissection of oxidative protein folding in yeast began with the isolation of an essential and conserved gene, ERO1 (ER oxidation), encoding a novel ER membrane protein required for protein oxi- dation in the ER 14,15 (Table 1). A temperature-sensitive allele of ERO1 was identified in a screen for mutants defective in the export from the ER of secretory pro- teins containing disulphide bonds 14 . Mutations in ERO1 were also isolated in a screen for S. cerevisiae strains with diminished oxidative capacity, a property reflected by increased sensitivity to the reductant dithiothreitol (DTT) 15 . Secretory proteins that would normally acquire intramolecular disulphide bonds remain completely reduced in the conditional ero1-1 mutant 14 . Ero1p appears to introduce oxidizing equivalents necessary for protein disulphide bond formation into the ER lumen, a conclusion supported by the observation that a membrane-permeable thiol oxidant can substitute for ERO1 function 14 . Moreover, overexpression of ERO1 increases the oxidative capacity of the cell 14,15 (Table 1). Recent findings indicate that Ero1p transfers di- sulphide bonds directly to Pdi1p. Although the REVIEWS Pathways for protein disulphide bond formation Alison R. Frand, John W. Cuozzo and Chris A. Kaiser The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems. The authors are in the Dept of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. E-mail: ckaiser@mit.edu