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

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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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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Correspondence to Tom A. Rapoport.

Supplementary information

SI Figure 1

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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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