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Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation

Nature Cell Biology volume 3, pages 24–29 (2001)Cite this article

Abstract

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin–proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin–protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro — catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.

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Figure 1: Functions of Hrd1p in vivo.
Figure 2: In vitro auto-ubiquitination of MBP–Hrd1p.
Figure 3: Hrd1 promotes transfer of ubiquitin to specific proteins in vitro .
Figure 4: HRD1 promotes ubiquitination by specific E2s.
Figure 5: HRD1 suppression of Cd2+ sensitivity.

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References

  1. Brodsky, J. L. & McCracken, A. A. ER protein quality control and proteasome-mediated protein degradation. Semin. Cell Dev. Biol. 10, 507–513 ( 1999).

    Article  CAS  Google Scholar 

  2. Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30, 405–439 (1996).

    Article  CAS  Google Scholar 

  3. Hampton, R. Y., Gardner, R. G. & Rine, J. Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol. Biol. Cell 7, 2029–2044 (1996).

    Article  CAS  Google Scholar 

  4. Wilhovsky, S. K., Gardner, R. G. & Hampton, R. Y. HRD gene dependence of ER-associated degradation. Mol. Biol. Cell 11, 1697– 1708 (2000).

    Article  CAS  Google Scholar 

  5. Friedlander, R., Jarosch, E., Urban, J., Volkwein, C. & Sommer, T. A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nature Cell Biol. 2, 379–384 (2000).

    Article  CAS  Google Scholar 

  6. Travers, K. J. et al. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101, 249–258 ( 2000).

    Article  CAS  Google Scholar 

  7. Lorick, K. L. et al. RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl Acad. Sci. USA 96, 11364–11369 (1999).

    Article  CAS  Google Scholar 

  8. Joazeiro, C. A. et al. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312 (1999).

    Article  CAS  Google Scholar 

  9. Ohta, T., Michel, J. J., Schottelius, A. J. & Xiong, Y. ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. Mol. Cell 3, 535–541 (1999).

    Article  CAS  Google Scholar 

  10. Nakanishi, M., Goldstein, J. L. & Brown, M. S. Multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme. J. Biol. Chem. 263, 8929–8937 (1988).

    CAS  PubMed  Google Scholar 

  11. Hampton, R. Y. & Rine, J. Regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum, in yeast. J. Cell Biol. 125, 299– 312 (1994).

    Article  CAS  Google Scholar 

  12. Hampton, R. Y. & Bhakta, H. Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase. Proc. Natl Acad. Sci. USA 94, 12944–12948 (1997).

    Article  CAS  Google Scholar 

  13. Gardner, R. G. & Hampton, R. Y. A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in eukaryotes. J. Biol. Chem. 274, 31671–31678 (1999).

    Article  CAS  Google Scholar 

  14. Cronin, S. R. & Hampton, R. Y. in Methods in Enzymology Vol. 302 (ed. Conn, P. M.) 58–73 (Academic Press, San Diego and London, 1999).

    Google Scholar 

  15. Bordallo, J., Plemper, R. K., Finger, A. & Wolf, D. H. Der3p–Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol. Biol. Cell 9, 209–222 ( 1998).

    Article  CAS  Google Scholar 

  16. Zachariae, W. et al. Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins. Science 279, 1216–1219 ( 1998).

    Article  CAS  Google Scholar 

  17. Seol, J. H. et al. Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev. 13, 1614–1626 (1999).

    Article  CAS  Google Scholar 

  18. Hiller, M. M., Finger, A., Schweiger, M. & Wolf, D. H. ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273, 1725– 1728 (1996).

    Article  CAS  Google Scholar 

  19. Sommer, T. & Jentsch, S. A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365, 176–179 ( 1993).

    Article  CAS  Google Scholar 

  20. Gardner, R. G. et al. ER degradation requires lumen to cytosol signaling: transmembrane control of Hrd1p by Hrd3p. J. Cell Biol. 151, 69–82 (2000).

    Article  CAS  Google Scholar 

  21. Richards, F. M. On the enzymic activity of subtilisin-modified ribonuclease. Proc. Natl Acad. Sci. USA 44, 162–166 (1958).

    Article  CAS  Google Scholar 

  22. Blackburn, P. & Moore, S. in The Enzymes (ed. Boyer, P. D.) 317–440 (Academic Press, San Diego, 1982).

    Google Scholar 

  23. Fang, S., Jensen, J. P., Ludwig, R. L., Vousden, K. H. & Weissman, A. M. Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J. Biol. Chem. 275, 8945–8951 (2000).

    Article  CAS  Google Scholar 

  24. Gonen, H. et al. Isolation, characterization, and partial purification of a novel ubiquitin-protein ligase, E3. Targeting of protein substrates via multiple and distinct recognition signals and conjugating enzymes. J. Biol. Chem. 271, 302–310 ( 1996).

    Article  CAS  Google Scholar 

  25. Hershko, A. & Heller, H. Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates. Biochem. Biophys. Res. Commun. 128, 1079–1086 ( 1985).

    Article  CAS  Google Scholar 

  26. Visintin, R., Prinz, S. & Amon, A. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science 278, 460–463 (1997).

    Article  CAS  Google Scholar 

  27. Gray, W. M. et al. Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana. Genes Dev. 13, 1678–1691 (1999).

    Article  CAS  Google Scholar 

  28. Kumar, S., Talis, A. L. & Howley, P. M. Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination. J. Biol. Chem. 274 , 18785–18792 (1999).

    Article  CAS  Google Scholar 

  29. Plemper, R. K., Egner, R., Kuchler, K. & Wolf, D. H. Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome. J. Biol. Chem. 273, 32848–32856 (1998).

    Article  CAS  Google Scholar 

  30. Seufert, W., McGrath, J. P. & Jentsch, S. UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation. EMBO J. 9, 4535–4541 ( 1990).

    Article  CAS  Google Scholar 

  31. Jungmann, J., Reins, H. A., Schobert, C. & Jentsch, S. Resistance to cadmium mediated by ubiquitin-dependent proteolysis. Nature 361, 369–371 ( 1993).

    Article  CAS  Google Scholar 

  32. Biederer, T., Volkwein, C. & Sommer, T. Role of Cue1p in ubiquitination and degradation at the ER surface. Science 278, 1806– 1809 (1997).

    Article  CAS  Google Scholar 

  33. Hill, K. & Cooper, A. A. Degradation of unassembled Vph1p reveals novel aspects of the yeast ER quality control system. EMBO J. 19, 550–561 ( 2000).

    Article  CAS  Google Scholar 

  34. Swerdlow, P. S., Finley, D. & Varshavsky, A. Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal. Biochem. 156, 147–153 (1986).

    Article  CAS  Google Scholar 

  35. Anan, T. et al. Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes . Genes Cells 3, 751–763 (1998).

    Article  CAS  Google Scholar 

  36. Young, I. T. Proof without prejudice: use of the Kolmogorov–Smirnov test for the analysis of histograms from flow systems and other sources. J. Histochem. Cytochem. 25, 935–941 (1977).

    Article  CAS  Google Scholar 

  37. Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355– 360 (1995).

    Article  CAS  Google Scholar 

  38. Kim, J. S. & Raines, R. T. Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci. 2, 348–356 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the American Heart Association, Western States Affiliate (to N.W.B.), ARCS Foundation, San Diego (to N.W.B.) and the National Institutes of Health (to R.Y.H.), and by a Searle Scholarship (to R.Y.H.).

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Authors and Affiliations

  1. Division of Biology, Section of Cell and Developmental Biology, University of California San Diego, 9500 Gilman Drive, La Jolla, 92093-0347, California, USA

    Nathan W. Bays, Richard G. Gardner, Linda P. Seelig & Randolph Y. Hampton

  2. Molecular Biology and Virology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, 92037-1099, California, USA

    Claudio A. Joazeiro

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Correspondence to Randolph Y. Hampton.

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Bays, N., Gardner, R., Seelig, L. et al. Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation. Nat Cell Biol 3, 24–29 (2001). https://doi.org/10.1038/35050524

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