contain E2 ubiquitin-conjugating enzymes and E3 ubiquitin- protein ligases that ubiquitylate the substrates as they emerge [2,4]. The heterodimeric cofactor Ufd1-Npl4 may simultaneously interact with p97 and ubiquitylated substrates [31,32] and assist p97 with substrate recruitment in ERAD [10] and other degradation pathways [33]. Then, peptide:N-glycanase associates with p97 and removes glycans from glycosylated ERAD substrates [34] before the substrate is finally transferred to the 26S proteasome and degraded. To broaden our understanding of p97 it is critical to obtain a more detailed knowledge of its various cofactors, of which at least 20 have been found so far [22,23]. Certainly, some functions of p97 are likely to be specific to certain cells or tissues. For instance, cells that secrete large amounts of protein, such as plasma and liver cells, probably have a highly active ERAD system and ERAD-relevant p97 cofactors may therefore be more abundant in such cells. However, so far no cell- or tissue specific p97 cofactors have been isolated or characterized. Here, we describe the tissue-specific human protein Rep8 (also known as Ubxd6), a previously cloned [35], but otherwise uncharacterized UBX protein present in higher eukaryotes. We found that Rep8 shares some similarity with the transmembrane p97 cofactor called VIMP [29]. Accordingly, we found that Rep8 is a transmem- brane protein, localized in the ER membrane. Rep8 associates with p97 via its cytoplasmic UBX domain to the N-domain of p97. In agreement with a previous report [35], we find that Rep8 is almost exclusively expressed in gonads. In the testis we find Rep8 expression in post-meiotic round spermatids. In ovaries Rep8 is expressed in granulosa cells. Knock-down of Rep8 expression results in delayed clearance of model ERAD substrates and reduced amounts of p97 associated with the ER membrane. Thus, like VIMP, Rep8 probably facilitates ERAD by tethering p97 to the ER membrane. Materials and Methods Buffers The buffers were: Buffer A, 25 mM Tris/HCl pH 7.5, 2 mM MgCl2, 2 mM ATP, 50 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100. Buffer B, 33 mM Hepes pH 7.3, 150 mM potassium acetate, 10% (v/v) glycerol, 1% (w/v) DeoxyBigChap (Fluka). Buffer C, 33 mM Hepes pH 7.3, 150 mM potassium acetate, 10% (v/v) glycerol, 0.2% (w/v) DeoxyBigChap (Fluka), 1 mg/mL BSA. Buffer D, 20 mM Hepes pH 7.5, 0.25 M sucrose, 1 mM DTT. Buffer E, 25 mM Tris/HCl pH 8.0, 500 mM NaCl, 1 mM DTT. Plasmids and expression For expression of recombinant Rep8, full-length cDNA and various truncations encoding human Rep8 were transferred to the appropriate Gateway destination vectors (Invitrogen). The expres- sion constructs for mouse p97 were kindly provided by Dr. Hemmo H. Meyer (Zurich, Switzerland). The proteins were expressed in E. coli BL21*(DE3) (Invitrogen) and purified by standard methods. The plasmid, used for expressing ER-targeted RFP, was generously supplied by Dr. Ulrike Kutay (Zurich, Switzerland). Yeast two-hybrid screening Yeast two-hybrid screening, using full length human p97, was performed on a HeLa cell cDNA library (Invitrogen) using the ProQuest yeast two-hybrid system (Invitrogen) according to the protocol, supplied by the manufacturer. Rep8 Is a Tissue-Specific p97 Cofactor Cell culture MelJuSo cells, stably transfected to express CD3d-YFP and ubiquitinG76V-YFP, were generously supplied by Dr. Nico P. Dantuma (Stockholm, Sweden). HeLa cells, stably transfected to express HA-tagged TCRa, were kindly supplied by Dr. Cezary Wo´ jcik (Evansville, Indiana). These cells and HeLa cells that were stably transfected to express Hrd1 with a biotin targeting sequence [25], were maintained in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 10% newborn- or fetal-calf serum (Invitrogen) at 37uC in a humidified atmosphere containing 5% CO2. Electrophoresis and blotting Proteins were separated on 7 cm68 cm 12.5% acrylamide gels. Proteins were transferred to BA83 (Schleicher & Schuell) nitrocellulose membranes by semi-dry blotting and probed with antibodies as indicated. Densitometry was performed using UnScanIt v.6.1 software (Silk Scientific). Purification of p97 from red blood cells Untagged p97 was purified from outdated human red blood cells by following a protocol for purification of 26S proteasomes [36]. After the final chromatographic step, fractions not containing 26S proteasomes were analyzed by dot blotting for the presence of p97. The p97-containing fractions were pooled and found by SDS-PAGE to contain pure p97. Antibodies Antibodies to human Rep8 were raised in rabbits by immunization with purified GST-tagged Rep8 residues 57–270, encompassing the entire cytoplasmic domain of Rep8. The antibodies to p97 have been described previously [37]. Antibodies to TMX3, calnexin, and ERp57 were generously supplied by Dr. Lars Ellgaard. Antibodies specific for proteasome subunits were from Enzo Life Sciences. Anti-pentaHis antibodies were pur- chased from Qiagen. The anti-GST, anti-GFP, anti-HA and anti- b-actin were purchased from Sigma. Peroxidase conjugated streptavidin was purchased from Dako. Assays The concentration of cell protein was determined using BCA (Pierce) or Bradford (Pierce) assays with BSA as a standard. Concentrations of purified recombinant proteins were determined from A280nm. Transfection Small interfering RNAs (HP GenomeWide siRNA SI04350934 and SI04323347), specific for human Rep8, were purchased from Qiagen. The siCONTROL siRNA#1 (Dharmacon) was used as an unspecific control. Exponentially growing MelJuSo or HeLa cells were washed in PBS and incubated for 24 h with 100 nM siRNA and 0.4% Dharmafect in DMEM supplemented with 1% calf serum. The medium was then changed to DMEM with 10% serum. The cultures were used after another 4 days. Co-precipitations Transformed E. coli BL21*(DE3) cells, expressing tagged protein, were lysed by sonication in one volume of buffer A. The extracts were cleared by centrifugation (12000 g, 30 min) and the fusion protein was purified. For precipitation experiments with HeLa cells stably transfected to express Hrd1 with a biotin tag, the cells were suspended in 4 volumes of buffer B and incubated with gentle agitation for 30 min. Cleared extracts were prepared by PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25061