On the subcellular level, Rep8, like VIMP [29], localizes to the ER- membrane. The Rep8 C-terminal UBX domain faces the cytoplasm and thus connects p97 to this organelle. Accordingly, when Rep8 is lacking less p97 is associated with the ER membrane. Conversely overexpression of Rep8 would presumably shift p97 to be more tightly connected with the ER membrane. However, unfortunately we were unable to overexpress Rep8 to a significant level. We found that upon knock-down of Rep8 expression, proteolysis of two model ERAD substrates was slightly retarded. We note that p97 plays other important roles at the ER- membrane, including transcription factor maturation [18] and membrane fusion [45], and we cannot rule out that the observed effect on ERAD is indirect and perhaps due to a perturbed p97 cofactor binding in response to the reduced amount of Rep8. Alterations in p97 cofactor binding may cause pleiotropic phenotypes and has been linked to disease [60]. As mentioned, Hrd1 may itself bind p97 directly, and it is therefore surprising that other p97 binding partners are needed to fulfill the same function. However, besides Hrd1 and Rep8, other transmembrane ERAD components such as VIMP [29], Erasin [30], and Derlin-1 [58] also bind p97 directly. This redundancy may explain why cells lacking Rep8 only display the moderate ERAD phenotype described here. Given the mild effect of Rep8 on the ERAD model substrates used here, another possibility is that Rep8 is involved in degradation of clients specific for reproductive tissue. Supporting Information Figure S1 Rep8 is a phylogenetically conserved protein in higher eukaryotes. Clustal W (v1.82) alignment of human (Hs) Rep8 with its bovine (Bt), mouse (Mm) and zebrafish (Br) orthologues. Identical and similar residues have been marked. The domain organization is indicated by the colored bars. Rep8 contains a signal sequence (red), a transmembrane domain (black), a region which is homologous to VIMP (blue), and a UBX domain (green). (TIF) References 1. Hiller MM, Finger A, Schweiger M, Wolf DH (1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273: 1725–1728. 2. Kincaid MM, Cooper AA (2007) ERADicate ER stress or die trying. Antioxid Redox Signal 9: 2373–2387. 3. Anelli T, Sitia R (2008) Protein quality control in the early secretory pathway. EMBO J 27: 315–327. 4. Nakatsukasa K, Brodsky JL (2008) The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum. Traffic 9: 861–870. 5. Lederkremer GZ (2009) Glycoprotein folding, quality control and ER-associated degradation. Curr Opin Struct Biol 19: 515–523. 6. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82: 373– 428. 7. 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Rabinovich E, Kerem A, Fro¨hlich KU, Diamant N, Bar-Nun S (2002) AAA- ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol 22: 626–634. Rep8 Is a Tissue-Specific p97 Cofactor Figure S2 Degradation of TCRa. Pulse-chase experiments performed on cells expressing HA-tagged TCRa. The cells were transfected with Rep8 siRNA#2 or control siRNA. At the indicated times during the chase period the substrate was retrieved by precipitation using antibodies specific for HA. The precipitated material was resolved by SDS-PAGE and visualized by phosphoi- maging. Slower migrating species (filled arrow) corresponding to glycosylated forms of the protein were visible. Knockdown of Rep8 expression caused a decrease in the degradation. (TIF) Figure S3 Rep8 does not affect degradation of a cytoplasmic proteasome substrate. Pulse-chase experiments were performed on MelJuSo cells expressing ubiquitin-G76V-YFP transfected with Rep8 siRNA#2 or control siRNA. At the indicated times during the chase period, ubiquitin-G76V-YFP was precipitated using antibodies specific for GFP. The precipi- tated material was resolved by SDS-PAGE and visualized by phosphoimaging. The asterisks (*) marks an unknown contami- nant. (TIF) Sequences of primers used for qRT-PCR. Table S1 (PDF) Acknowledgments We thank Dr. Nico P. Dantuma, Dr. Lars Ellgaard, Dr. Ulrike Kutay, Dr. Hemmo H. Meyer and Dr. Cezary Wo´jcik for sharing valuable reagents and Anne-Marie Lauridsen, Dr. Doris Roth and Dr. Janos Steffen for technical assistance. We thank Dr. Klavs B. Hendil for helpful discussions and comments on the manuscript. Author Contributions Conceived and designed the experiments: LM IRA RHP. Performed the experiments: LM FK AV DB SP. Analyzed the data: LM IRA RHP. Contributed reagents/materials/analysis tools: MS KH. 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Cell 134: 804–816. PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25061