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The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol

Nature volume 414, pages 652–656 (2001)Cite this article

Abstract

In eukaryotic cells, incorrectly folded proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded by the proteasome1. This pathway is co-opted by some viruses. For example, the US11 protein of the human cytomegalovirus targets the major histocompatibility complex class I heavy chain for cytosolic degradation2. How proteins are extracted from the ER membrane is unknown. In bacteria and mitochondria, members of the AAA ATPase family are involved in extracting and degrading membrane proteins3,4. Here we demonstrate that another member of this family, Cdc48 in yeast and p97 in mammals, is required for the export of ER proteins into the cytosol. Whereas Cdc48/p97 was previously known to function in a complex with the cofactor p47 (ref. 5) in membrane fusion6,7,8, we demonstrate that its role in ER protein export requires the interacting partners Ufd1 and Npl4. The AAA ATPase interacts with substrates at the ER membrane and is needed to release them as polyubiquitinated species into the cytosol. We propose that the Cdc48/p97–Ufd1–Npl4 complex extracts proteins from the ER membrane for cytosolic degradation.

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Figure 1: Cdc48 is required for ER protein degradation in S. cerevisiae.
Figure 2: The Cdc48 interacting proteins Ufd1 and Npl4 are required for ER protein degradation.
Figure 3: Mammalian p97 is required for export of MHC class I heavy chains from the ER.
Figure 4: Association of p97 with MHC class I heavy chains.

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References

  1. Bonifacino, J. S. & Weissman, A. M. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell Dev. Biol. 14, 19–57 (1998).

    Article  CAS  Google Scholar 

  2. Wiertz, E. J. et al. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384, 432–438 (1996).

    Article  ADS  CAS  Google Scholar 

  3. Kihara, A., Akiyama, Y. & Ito, K. Dislocation of membrane proteins in FtsH-mediated proteolysis. EMBO J. 18, 2970–2981 (1999).

    Article  CAS  Google Scholar 

  4. Leonhard, K. et al. Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface. Mol. Cell 5, 629–638 (2000).

    Article  CAS  Google Scholar 

  5. Kondo, H. et al. p47 is a cofactor for p97-mediated membrane fusion. Nature 388, 75–78 (1997).

    Article  CAS  Google Scholar 

  6. Latterich, M., Fröhlich, K. U. & Schekman, R. Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell 82, 885–893 (1995).

    Article  CAS  Google Scholar 

  7. Rabouille, C., Levine, T. P., Peters, J. M. & Warren, G. An NSF-like ATPase, p97 and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell 82, 905–914 (1995).

    Article  CAS  Google Scholar 

  8. Acharya, U. et al. The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion events. Cell 82, 895–904 (1995).

    Article  CAS  Google Scholar 

  9. Ghislain, M., Dohmen, R., 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 

  10. Koegl, M. et al. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644 (1999).

    Article  CAS  Google Scholar 

  11. Hoppe, T. et al. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing. Cell 102, 577–586 (2000).

    Article  CAS  Google Scholar 

  12. 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 

  13. DeHoratius, C. & Silver, P. A. Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene. Mol. Biol. Cell 7, 1835–1855 (1996).

    Article  CAS  Google Scholar 

  14. Hitchcock, A. L. et al. The conserved Npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation. Mol. Biol. Cell 12, 3226–3241 (2001).

    Article  CAS  Google Scholar 

  15. Dai, R., Chen, E., Longo, D. L., Gorbea, C. M. & Li, C. C. Involvement of valosin-containing protein, an ATPase co-purified with Iκbα and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IκBα. J. Biol. Chem. 273, 3562–3573 (1998).

    Article  CAS  Google Scholar 

  16. Verma, R. et al. Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell 11, 3425–3439 (2000).

    Article  CAS  Google Scholar 

  17. Casagrande, R. et al. Degradation of proteins from the ER of S. cerevisiae requires an intact unfolded protein response pathway. Mol. Cell 5, 729–735 (2000).

    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  ADS  CAS  Google Scholar 

  19. 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 

  20. Shamu, C. E., Story, C. M., Rapoport, T. A. & Ploegh, H. L. The pathway of US11-dependent degradation of MHC class I heavy chains involves a ubiquitin-conjugated intermediate. J. Cell Biol. 147, 45–57 (1999).

    Article  CAS  Google Scholar 

  21. Lamb, J. R., Fu, V., Wirtz, E. & Bangs, J. D. Functional analysis of the trypanosomal AAA protein TbVCP with Trans-dominant ATP hydrolysis mutants. J. Biol. Chem. 276, 21512–21520 (2001).

    Article  CAS  Google Scholar 

  22. Meyer, H. H., Kondo, H. & Warren, G. The p47 co-factor regulates the ATPase activity of the membrane fusion protein, p97. FEBS Lett. 437, 255–257 (1998).

    Article  CAS  Google Scholar 

  23. Shamu, C. E., Flierman, D., Ploegh, H. L., Rapoport, T. A. & Chau, V. Polyubiquitination is required for US11-dependent movement of MHC class I heavy chain from the ER into the cytosol. Mol. Biol. Cell 12, 2546–2555 (2001).

    Article  CAS  Google Scholar 

  24. Dai, R. & Li, C. C. Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nature Cell Biol. 3, 740–744 (2001).

    Article  CAS  Google Scholar 

  25. Zhang, X. et al. Structure of the AAA ATPase p97. Mol. Cell 6, 1473–1484 (2000).

    Article  CAS  Google Scholar 

  26. Singleton, M. R., Sawaya, M. R., Ellenberger, T. & Wigley, D. B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000).

    Article  CAS  Google Scholar 

  27. Schmidt, M., Lupas, A. N. & Finley, D. Structure and mechanism of ATP-dependent proteases. Curr. Opin. Chem. Biol. 3, 584–591 (1999).

    Article  CAS  Google Scholar 

  28. Matlack, K. E., Misselwitz, B., Plath, K. & Rapoport, T. A. BiP acts as a molecular ratchet during posttranslational transport of Prepro-α factor across the ER membrane. Cell 97, 553–564 (1999).

    Article  CAS  Google Scholar 

  29. Thuret, J., Valay, J., Faye, G. & Mann, C. Civ1 (CAK in vivo), a novel Cdk-activating kinase. Cell 86, 565–576 (1996).

    Article  CAS  Google Scholar 

  30. Deshaies, R. J. & Schekman, R. A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J. Cell Biol. 105, 633–645 (1987).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Shamu, R. Casagranda, A. Hitchcock and N. Bouquin for discussions; D. Schoffnegger for experimental help; C. Shamu, W. Prinz, B. Tsai, D. Flierman, B. DeDecker and D. Finley for critical reading of the manuscript; and G. Warren for support and comments. Y.Y. is supported by the Helen Hay Whitney postdoctoral fellowship. H.M. was supported by the National Institutes of Health (NIH) and Human Frontier Science Program grants and T.A.R. by an NIH grant. T.A.R. is a Howard Hughes Medical Institute Investigator.

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Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Yihong Ye & Tom A. Rapoport

  2. Department of Cell Biology, Yale Medical School, 333 Cedar Street, SHM C432, New Haven, 06520, Connecticut, USA

    Hemmo H. Meyer

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  1. Yihong Ye
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Correspondence to Tom A. Rapoport.

Supplementary information

SI Figure 1

(JPG 15.35 KB)

Stability of CPY* in various ufd mutants. The stability of CPY* in various ufd deletion mutants and in an isogenic wild type strain was analyzed by immunoblotting after addition of cycloheximide.

SI Figure 2

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Export of CPY* from the ER is inhibited in cdc48, ufd1 and npl4 mutants. a, Wild type or mutant cells were incubated with cycloheximide for the indicated time periods. Spheroplasts were prepared and homogenized. Aliquots of the homogenate were centrifuged to obtain membrane pellet (P) and supernatant (S) fractions. These were analyzed by immunoblotting with antibodies to CPY and Sec61p. b, Homogenates from a wild type strain, prepared as in a, were incubated with proteinase K in the absence or presence of Triton X-100. The samples were analyzed by immunoblotting with antibodies to the indicated proteins. c, Homogenates from wild type or mutant cells that were incubated with cycloheximide for the indicated time periods were treated with proteinase K as in b. The samples were analyzed by immunoblotting with CPY antibodies.

SI Figure 3

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Purification and characterization of wild type and mutant p97. a, Wild type (wt) or mutant (E305/578Q) (QQ) p97 were expressed in E. coli as His-tagged proteins and purified. Shown is a SDS-PAGE stained with Coomassie blue (2 ug protein loaded). For comparison, the same amount of purified rat liver p97 was loaded. b, Wild type or mutant p97 (QQ) were incubated with GST, GST-Ufd1/Npl4, or GST-p47. The GST-fusion proteins were bound to glutathione beads and analyzed for p97 binding by SDS-PAGE and staining with Coomassie blue. The lower panel shows 10% of the input material. c, The ATPase activities of rat liver p97 and recombinant wild type and mutant p97 were determined. The data represent the mean +/-SEM (n=3).

SI Figure 4

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Quantitation of endogenous p97. Control or US11 cells were permeabilized and 10ul of the cytosol fraction, corresponding to 1.5 x 105 cells, were analyzed by SDS-PAGE and immunoblotting with anti-p97 antibodies. Different amounts of p97 (wt) or p97 (QQ) were analyzed in parallel. The data indicate that 10ul of cytosol contains between 0.125-0.25ug p97.

SI Figure 5

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The release of poly-ubiquitinated heavy chains into the cytosol is inhibited by p47. US11 cells were labeled, permeabilized, and treated with 6ug p47 per 1X106 cells. After fractionation into a membrane pellet (P) and cytosol (S) fraction, the samples were sequentially immunoprecipitated with heavy chain and ubiquitin antibodies. A non-fractionated sample (T) was analyzed in parallel.

SI Figure 6

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Interaction of p97 with HA-tagged MHC class I heavy chains. Cells expressing both US11 and HA-tagged MHC heavy chain (HA/A2) were labeled with [35S]-methionine and permeabilized in the presence or absence of His-tagged wild type p97. Where indicated, ATP was depleted. The samples were then warmed up and incubated for the indicated time period (chase). Sequential immunoprecipitation with His and HA antibodies was carried out. HC+CHO and HC-CHO indicate glycosylated and deglycosylated heavy chain

SI Figure 7

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The interaction of p97 with heavy chains occurs prior to solubilization of the membranes. US11 cells were labeled and permeabilized. His-tagged wild type p97 was added either before the samples were chase-incubated, separated into membrane pellet (P) and supernatant (S) fractions, and detergent was added (before sol.), or it was added only after the addition of detergent (after sol.). Immunoprecipitation was carried out with either heavy chain (anti-HC) (top panel) or sequentially with His (anti-His) and heavy chain antibodies (bottom panel). A separate aliquot was immunoprecipitated directly with His antibodies and analyzed by Coomassie staining (middle panel).

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Ye, Y., Meyer, H. & Rapoport, T. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652–656 (2001). https://doi.org/10.1038/414652a

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