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Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation

Abstract

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that removes misfolded proteins from the ER1,2. ERAD substrates are channelled from the ER via a proteinacious pore to the cytosolic ubiquitin–proteasome system — a process involving dedicated ubiquitin ligases3,4,5 and the chaperone-like AAA ATPase Cdc48 (also known as p97)6,7,8,9,10. How the activities of these proteins are coupled remains unclear. Here we show that the UBX domain protein Ubx2 is an integral ER membrane protein that recruits Cdc48 to the ER. Moreover, Ubx2 mediates binding of Cdc48 to the ubiquitin ligases Hrd1 and Doa10, and to ERAD substrates. In addition, Ubx2 and Cdc48 interact with Der1 and Dfm1, yeast homologues of the putative dislocation pore protein Derlin-1 (refs 1113). Lack of Ubx2 causes defects in ERAD that are exacerbated under stress conditions. These findings are consistent with a model in which Ubx2 coordinates the assembly of a highly efficient ERAD machinery at the ER membrane.

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Figure 1: Ubx2 is an integral membrane protein of the ER.
Figure 2: Ubx2 recruits Cdc48 and Ufd1 to the ER and is able to assemble into a Cdc48Ufd1–Npl4–Ubx2 complex.
Figure 3: Ubx2 recruits Cdc48 to ERAD substrates and ERAD components.
Figure 4: Ubx2 functions in ERAD.
Figure 5: Ubx2 coordinates multiple interactions along the ERAD pathway.

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References

  1. Hampton, R. Y. ER-associated degradation in protein quality control and cellular regulation. Curr. Opin. Cell Biol. 14, 476–482 (2002).

    Article  CAS  Google Scholar 

  2. Kostova, Z. & Wolf, D. H. For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection. EMBO J. 22, 2309–2317 (2003).

    Article  CAS  Google Scholar 

  3. Bays, N. W., Gardner, R. G., Seelig, L. P., Joazeiro, C. A. & Hampton, R. Y. Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation. Nature Cell Biol. 3, 24–29 (2001).

    Article  CAS  Google Scholar 

  4. Deak, P. M. & Wolf, D. H. Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation. J. Biol. Chem. 276, 10663–10669 (2001).

    Article  CAS  Google Scholar 

  5. Swanson, R., Locher, M. & Hochstrasser, M. A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matα2 repressor degradation. Genes Dev. 15, 2660–2674 (2001).

    Article  CAS  Google Scholar 

  6. Bays, N. W., Wilhovsky, S. K., Goradia, A., Hodgkiss-Harlow, K. & Hampton, R. Y. HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol. Biol. Cell 12, 4114–4128 (2001).

    Article  CAS  Google Scholar 

  7. Ye, Y., Meyer, H. H. & Rapoport, T. A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652–656 (2001).

    Article  CAS  Google Scholar 

  8. Braun, S., Matuschewski, K., Rape, M., Thoms, S. & Jentsch, S. Role of the ubiquitin-selective CDC48(UFD1/NPL4)chaperone (segregase) in ERAD of OLE1 and other substrates. EMBO J. 21, 615–621 (2002).

    Article  CAS  Google Scholar 

  9. Jarosch, E. et al. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nature Cell Biol. 4, 134–139 (2002).

    Article  CAS  Google Scholar 

  10. Rabinovich, E., Kerem, A., Frohlich, K. U., Diamant, N. & Bar-Nun, S. AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol. Cell. Biol. 22, 626–634 (2002).

    Article  CAS  Google Scholar 

  11. Hitt, R. & Wolf, D. H. Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins. FEMS Yeast Res. 4, 721–729 (2004).

    Article  CAS  Google Scholar 

  12. Lilley, B. N. & Ploegh, H. L. A membrane protein required for dislocation of misfolded proteins from the ER. Nature 429, 834–840 (2004).

    Article  CAS  Google Scholar 

  13. Ye, Y., Shibata, Y., Yun, C., Ron, D. & Rapoport, T. A. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429, 841–847 (2004).

    Article  CAS  Google Scholar 

  14. Woodman, P. G. p97, a protein coping with multiple identities. J. Cell Sci. 116, 4283–4290 (2003).

    Article  CAS  Google Scholar 

  15. Zhang, X., Beuron, F. & Freemont, P. S. Machinery of protein folding and unfolding. Curr. Opin. Struct. Biol. 12, 231–238 (2002).

    Article  Google Scholar 

  16. Johnson, E. S., Ma, P. C., Ota, I. M. & Varshavsky, A. A proteolytic pathway that recognizes ubiquitin as a degradation signal. J. Biol. Chem. 270, 17442–17456 (1995).

    Article  CAS  Google Scholar 

  17. Ghislain, M., Dohmen, R. J., Levy, F. & Varshavsky, A. Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae. EMBO J. 15, 4884–4899 (1996).

    Article  CAS  Google Scholar 

  18. Rape, M. et al. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone. Cell 107, 667–677 (2001).

    Article  CAS  Google Scholar 

  19. Buchberger, A., Howard, M. J., Proctor, M. & Bycroft, M. The UBX domain: a widespread ubiquitin-like module. J. Mol. Biol. 307, 17–24 (2001).

    Article  CAS  Google Scholar 

  20. Buchberger, A. From UBA to UBX: new words in the ubiquitin vocabulary. Trends Cell Biol. 12, 216–221 (2002).

    Article  CAS  Google Scholar 

  21. Decottignies, A., Evain, A. & Ghislain, M. Binding of Cdc48p to a ubiquitin-related UBX domain from novel yeast proteins involved in intracellular proteolysis and sporulation. Yeast 21, 127–139 (2004).

    Article  CAS  Google Scholar 

  22. Hartmann-Petersen, R. et al. The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes. Curr. Biol. 14, 824–828 (2004).

    Article  CAS  Google Scholar 

  23. Schuberth, C., Richly, H., Rumpf, S. & Buchberger, A. Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation. EMBO Rep. 5, 818–824 (2004).

    Article  CAS  Google Scholar 

  24. Wang, Y., Satoh, A., Warren, G. & Meyer, H. H. VCIP135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic Golgi fragments. J. Cell Biol. 164, 973–978 (2004).

    Article  CAS  Google Scholar 

  25. Wojcik, C., Yano, M. & DeMartino, G. N. RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J. Cell Sci. 117, 281–292 (2004).

    Article  CAS  Google Scholar 

  26. Meyer, H. H., Shorter, J. G., Seemann, J., Pappin, D. & Warren, G. A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, 2181–2192 (2000).

    Article  CAS  Google Scholar 

  27. 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 

  28. 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 

  29. Knop, M., Finger, A., Braun, T., Hellmuth, K. & Wolf, D. H. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J. 15, 753–763 (1996).

    Article  CAS  Google Scholar 

  30. Vashist, S. & Ng, D. T. Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J. Cell Biol. 165, 41–52 (2004).

    Article  CAS  Google Scholar 

  31. Finley, D., Ozkaynak, E. & Varshavsky, A. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48, 1035–1046 (1987).

    Article  CAS  Google Scholar 

  32. Wach, A. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12, 259–265 (1996).

    Article  CAS  Google Scholar 

  33. Knop, M. et al. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15, 963–972 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Jentsch for continued support and for providing yeast strains, plasmids and antibodies; D. Ng for providing plasmids for the expression of ERAD substrates; M. Knop for providing p415-GAL1-eGFP; M. Seedorf for providing anti-Kar2; T. Sommer for providing antibodies and for helpful discussions; S. Köglsberger for technical assistance; S. Jentsch, S. Müller, S. Braun and S. Rumpf for critical reading of the manuscript and helpful discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Emmy Noether grant Bu 951/1-3) and the German-Israeli Foundation for Scientific Research and Development (Young Scientists' grant 2049) to A.B.

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Correspondence to Alexander Buchberger.

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Schuberth, C., Buchberger, A. Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation. Nat Cell Biol 7, 999–1006 (2005). https://doi.org/10.1038/ncb1299

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