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A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation

Nature Cell Biology volume 3, pages 874–882 (2001)Cite this article

Abstract

Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol–disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway.

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Figure 1: ERV2 overexpression restores growth, resistance to DTT, and the formation of disulphide bonds to an ero1-1 mutant.
Figure 2: Erv2p is a membrane protein located in the ER.
Figure 3: Erv2p contains a conserved domain shared between yeast and human proteins.
Figure 4: Erv2p is a flavin-binding thiol oxidase that consumes oxygen to form disulphide bonds.
Figure 5: Erv2p uses oxygen in a pathway for thiol oxidation in vivo.
Figure 6: Erv2p transfers oxidizing equivalents to Pdi1p via a direct thiol–disulphide exchange.
Figure 7: Pathways for thiol oxidation in the ER.

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References

  1. Lyles, M. M. & Gilbert, H. F. Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry 30, 613–619 (1991).

    Article  CAS  Google Scholar 

  2. Frand, A. R. & Kaiser, C. A. The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum. Mol. Cell 1, 161–170 (1998).

    Article  CAS  Google Scholar 

  3. Pollard, M. G., Travers, K. J. & Weissman, J. S. Ero1p: a novel and ubiquitous protein with an essential role in oxidative protein folding in the endoplasmic reticulum. Mol. Cell 1, 171–182 (1998).

    Article  CAS  Google Scholar 

  4. Cuozzo, J. W. & Kaiser, C. A. Competition between glutathione and protein thiols for disulphide-bond formation. Nature Cell Biol. 1, 130–135 (1999).

    Article  CAS  Google Scholar 

  5. Cabibbo, A. et al. ERO1-L, a human protein that favors disulfide bond formation in the endoplasmic reticulum. J. Biol. Chem. 275, 4827–4833 (2000).

    Article  CAS  Google Scholar 

  6. Gilbert, H. F. in Mechanisms of Protein Folding (ed. Pain, R. H.) 104–135 (Oxford Univ. Press, New York, 1994).

    Google Scholar 

  7. Chivers, P. T., Prehoda, K. E. & Raines, R. T. The CXXC motif: a rheostat in the active site. Biochemistry 36, 4061–4066 (1997).

    Article  CAS  Google Scholar 

  8. Martin, J. L. Thioredoxin—a fold for all reasons. Structure 3, 245–250 (1995).

    Article  CAS  Google Scholar 

  9. Scherens, B., Dubois, E. & Messenguy, F. Determination of the sequence of the yeast YCL313 gene localized on chromosome III. Homology with the protein disulfide isomerase (PDI gene product) of other organisms. Yeast 7, 185–193 (1991).

    Article  CAS  Google Scholar 

  10. Holst, B., Tachibana, C. & Winther, J. R. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum. J. Cell Biol. 138, 1229–1238 (1997).

    Article  CAS  Google Scholar 

  11. Frand, A. R. & Kaiser, C. A. Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum. Mol. Cell 4, 469–477 (1999).

    Article  CAS  Google Scholar 

  12. Laboissiere, M. C., Sturley, S. L. & Raines, R. T. The essential function of protein-disulfide isomerase is to unscramble non-native disulfide bonds. J. Biol. Chem. 270, 28006–28009 (1995).

    Article  CAS  Google Scholar 

  13. LaMantia, M. L. & Lennarz, W. J. The essential function of yeast protein disulfide isomerase does not reside in its isomerase activity. Cell 74, 899–908 (1993).

    Article  CAS  Google Scholar 

  14. Frand, A. R., Cuozzo, J. W. & Kaiser, C. A. Pathways for protein disulphide bond formation. Trends Cell Biol. 10, 203–210 (2000).

    Article  CAS  Google Scholar 

  15. Tu, B. P., Ho-Schleyer, S. C., Travers, K. J. & Weissman, J. S. Biochemical basis of oxidative folding in the endoplasmic reticulum. Science 290, 1571–1574 (2000).

    Article  CAS  Google Scholar 

  16. Stein, G. & Lisowsky, T. Functional comparison of the yeast scERV1 and scERV2 genes. Yeast 14, 171–180 (1998).

    Article  CAS  Google Scholar 

  17. Jämsä, E., Simonen, M. & Makarow, M. Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent. Yeast 10, 355–370 (1994).

    Article  Google Scholar 

  18. Simons, J. F., Ferro-Novick, S., Rose, M. D. & Helenius, A. BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast. J. Cell Biol. 130, 41–49 (1995).

    Article  CAS  Google Scholar 

  19. Lisowsky, T. Dual function of a new nuclear gene for oxidative phosphorylation and vegetative growth in yeast. Mol. Gen. Genet. 232, 58–64 (1992).

    Article  CAS  Google Scholar 

  20. Hofhaus, G., Stein, G., Polimeno, L., Francavilla, A. & Lisowsky, T. Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization. Eur. J. Cell Biol. 78, 349–356 (1999).

    Article  CAS  Google Scholar 

  21. Francavilla, A. et al. Augmenter of liver regeneration: its place in the universe of hepatic growth factors. Hepatology 20, 747–757 (1994).

    Article  CAS  Google Scholar 

  22. Hagiya, M. et al. Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution. Proc. Natl Acad. Sci. USA 91, 8142–8146 (1994).

    Article  CAS  Google Scholar 

  23. Coppock, D. L., Cina-Poppe, D. & Gilleran, S. The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERV1. Genomics 54, 460–468 (1998).

    Article  CAS  Google Scholar 

  24. Lewis, T., Zsak, L., Burrage, T. G., Lu, Z., Kutish, G. F., Neilan, J. G. & Rock, D. L. An African swine fever virus ERV1-ALR homologue, 9GL, affects virion maturation and viral growth in macrophages and viral virulence in swine. J. Virol. 74, 1275–1285 (2000).

    Article  CAS  Google Scholar 

  25. Senkevich, T. G., White, C. L., Koonin, E. V. & Moss, B. A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation. Proc. Natl Acad. Sci. USA 97, 12068–12073 (2000).

    Article  CAS  Google Scholar 

  26. Hoober, K. L., Glynn, N. M., Burnside, J., Coppock, D. L. & Thorpe, C. Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases. J. Biol. Chem. 274, 31759–31762 (1999).

    Article  CAS  Google Scholar 

  27. Leea, J., Hofhausa, G. & Lisowsky, T. Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase. FEBS Lett. 477, 62–66 (2000).

    Article  Google Scholar 

  28. Faeder, E. J. & Siegel, L. M. A rapid micromethod for determination of FMN and FAD in mixtures. Anal. Biochem. 53, 332–336 (1973).

    Article  CAS  Google Scholar 

  29. Claros, M. G. & von Heijne, G. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10, 685–686 (1994).

    CAS  PubMed  Google Scholar 

  30. Gerber, J., Mühlenhoff, U., Hofhaus, G., Lill, R. & Lisowsky, T. Yeast Erv2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family. J. Biol. Chem. 276, 23486–23489 (2001).

    Article  CAS  Google Scholar 

  31. Hirschberg, C. B., Robbins, P. W. & Abeijon, C. Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu. Rev. Biochem. 67, 49–69 (1998).

    Article  CAS  Google Scholar 

  32. Gliszczynska, A. & Koziolowa, A. Chromatographic determination of flavin derivatives in baker's yeast. J. Chromatogr. A 822, 59–66 (1998).

    Article  CAS  Google Scholar 

  33. Pedrajas, J. R., Kosmidou, E., Miranda-Vizuete, A., Gustafsson, J. A., Wright, A. P. & Spyrou, G. Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae. J. Biol. Chem. 274, 6366–6373 (1999).

    Article  CAS  Google Scholar 

  34. Lisowsky, T., Lee, J. E., Polimeno, L., Francavilla, A. & Hofhaus, G. Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase. Dig. Liver Dis. 33, 173–180 (2001).

    Article  CAS  Google Scholar 

  35. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).

    Article  CAS  Google Scholar 

  36. Liu, H., Krizek, J. & Bretscher, A. Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast. Genetics 132, 665–673 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Elrod-Erickson, M. J. & Kaiser, C. A. Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations. Mol. Biol. Cell 7, 1043–1058 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Winther for generously providing the PDI expression plasmid, and T. Stevens for PDI antibody. Support for this work came from a grant from the National Institutes of General Medical Sciences. J.W.C. was supported by a NIH National Research Service Award, C.S.S. was supported by a NIH National Research Service Award, and A.V. was supported by a fellowship from the Gulbenkian Foundation.

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  1. John W. Cuozzo

    Present address: Genetics Institute, Wyeth Research, 87 Cambridge Park Dr., Cambridge, Massachusetts, 02140, USA

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  1. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Carolyn S. Sevier, John W. Cuozzo, Andrea Vala, Fredrik Åslund & Chris A. Kaiser

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Correspondence to Chris A. Kaiser.

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Sevier, C., Cuozzo, J., Vala, A. et al. A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation. Nat Cell Biol 3, 874–882 (2001). https://doi.org/10.1038/ncb1001-874

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