Trends in Cell Biology
Volume 10, Issue 5, 1 May 2000, Pages 203-210
Journal home page for Trends in Cell Biology

Review
Pathways for protein disulphide bond formation

https://doi.org/10.1016/S0962-8924(00)01745-1Get rights and content

Abstract

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.

Section snippets

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 oxidation in the ER14, 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 proteins containing disulphide bonds14. Mutations in ERO1 were also isolated in a screen for S. cerevisiae strains with diminished

The role of glutathione in oxidative protein folding in the ER

The thiol–disulphide redox status of intralumenal glutathione has long been the focus of considerations of how relatively oxidizing conditions are established within the ER12. Glutathione is the major small-molecule redox buffer in the ER, and the ratio of the concentration of GSH to GSSG in the ER (1:1 to 3:1) is similar to that found in redox buffers affording optimal rates for oxidative refolding in vitro12. From these, observations, it was natural to suppose that GSSG serves as the primary

Similarities in eukaryotic and prokaryotic disulphide-bond-forming pathways

The pathway for protein disulphide bond formation in the bacterial periplasm provides a useful analogy for the protein oxidation system in eukaryotes. Two enzymes drive disulphide bond formation in periplasmic proteins: the thioredoxin-like thiol–disulphide oxidoreductase DsbA and the cytoplasmic membrane protein DsbB29, 30 (Table 1). The active-site cysteines of DsbA form a disulphide bond that is transferred directly to periplasmic proteins31, after which DsbA is efficiently re-oxidized by

The family of PDI homologues

Several oxidoreductases homologous to PDI are found in the ER of both yeast and mammalian cells (Table 2). These PDI homologues have been implicated in diverse processes including not only oxidative protein folding but also the assembly of multiprotein complexes and the recognition of misfolded proteins in the ER39. Erp57 and Erp72 are mammalian homologues of PDI induced under conditions of ER stress39 (Table 2). Yeast homologues of PDI expressed in the ER lumen are Mpd1p, Mpd2p, Eug1p and Eps1p

Models for disulphide bond isomerization in eukaryotes

The similarities observed thus far between eukaryotic and prokaryotic disulphide-bond-forming systems suggest that commonalities might also be found between disulphide bond isomerization pathways in eukaryotes and the DsbC–DsbD system in prokaryotes. Eukaryotic secretory proteins typically contain more disulphide bonds than their prokaryotic counterparts, indicating that there might be an even greater need for disulphide bond reducing or reshuffling functions in the ER than in the periplasm.

Where do the oxidizing equivalents come from?

Although Ero1p appears to be the key conduit for introducing oxidizing equivalents into the ER lumen, it remains unclear how Ero1p itself is re-oxidized. Because Ero1p engages in thiol–disulphide exchange with Pdi1p, the identification of one or more redox-active cysteine pairs is anticipated in Ero1p. These active-site cysteines might be found amongst the seven conserved cysteine residues of Ero1p, three of which appear in the sequence Cys-x-x-Cys-x-x-Cys near the C-terminus of Ero1p14, 15.

Summary and future prospects

The work reviewed here places Ero1p and Pdi1p in an enzymatic pathway for protein disulphide bond formation in the ER16 and demonstrates that intralumenal GSH competes with protein thiols for oxidizing equivalents derived from Ero1p25. These studies provide a solid framework for further genetic and biochemical analysis of oxidative protein folding in eukaryotes. It will be of great interest to see whether analogies between eukaryotic and prokaryotic systems continue to hold as more details of

Acknowledgements

We are grateful to Peter Chivers and Fredrik Åslund for comments on this manuscript. We apologize to those authors whose work we could not cite directly owing to space limitations. Work in the laboratory of the authors was supported by grants from the National Institute of General Medical Sciences (to C.A.K.), an NIH predoctoral traineeship (to A.R.F.) and an NIH National Research Service Award (to J.W.C.).

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