centrifugation as above. Aliquots of extracts were incubated at 4uC for 4 h with 20 mL glutathione Sepharose beads (GE Healthcare) loaded with GST or the GST-tagged proteins. The beads were washed in 3615 mL of buffer A for recombinant proteins or buffer C for HeLa cell extracts. The precipitation experiments with purified p97 were performed in buffer A using about 3 mg p97 per reaction. Bound proteins were analyzed by SDS-PAGE and immunoblotting. Differential centrifugation Differential centrifugation for subcellular localization of Rep8 was performed essentially as described [38]. To quantify the amount of p97 associated with cell membranes, dilution series of the appropriate fractions were quantified by SDS-PAGE and immunoblotting. Membrane topology The orientation of Rep8 in the ER membrane was determined by Proteinase K and Triton X-100 treatment of microsomes. Briefly, HeLa cells were harvested and lysed in buffer D by passing through a 27 gauge needle. Unbroken cells were removed by centrifugation (300 g, 3 min) and the membrane fraction isolated by centrifugation (100000 g, 1 h). The membranes were resus- pended in buffer E. Aliquots of the membrane fraction were treated on ice with 1 mg/mL proteinase K (Sigma) and/or 1% Triton X-100 for 1 h. PMSF was then added to 5 mM before the samples were subjected to TCA precipitation. The precipitates were washed with ice cold acetone, resuspended in SDS sample buffer and resolved by SDS-PAGE and immunoblotting. Pulse-chase experiments The stability of the model substrates was followed by pulse- chase analysis of transfectants stably expressing the substrate, as described previously [39]. Fluorescence microscopy For fluorescence microscopy, HeLa cells were transiently co- transfected with plasmids for expressing Rep8 with a C-terminal GFP-tag and RFP modified to contain a signal sequence and the ER retention sequence, KDEL. About 48 h after transfection, the cells were fixed with 4% formaldehyde in PBS and mounted for confocal microscopy as described previously [25]. In situ hybridization Bases 1–559 of mouse Rep8 cDNA (Genbank sequence accession BC024492) from an IMAGE full-length cDNA plasmid (ImaGenes) were subcloned into pCMV-SPORT6, and used to generate sense and anti-sense digoxigenin-labelled Rep8 RNA probes by in vitro transcription. In situ hybridization to Bouin’s fixed adult mouse ovary and testis sections was performed as described [40]. Bound probe was visualized with NBT/BCIP substrate (Vector Laboratories) and sections counterstained with nuclear fast red. qRT-PCR Oligo dT-primed cDNA was prepared from testes of prepu- bertal or adult Tex19.12/2 and littermate control mice as described [41]. Sequences of primers, used for qRT-PCR, are listed in the supplementary material (Table S1). 20 mL of qPCRs containing 250 nM of each primer and 16 Brilliant II SYBR green master mix (Stratagene) were set up in triplicate and run on a Bio-Rad C1000 thermal cycler equipped with a CFX96 real time system. Primer pairs were validated as amplifying at 95– Rep8 Is a Tissue-Specific p97 Cofactor 100% efficiency, and expression levels were calculated relative to b-actin, using the 22DDCT method. No significant qPCR amplification was detected in control cDNAs generated in the absence of reverse transcriptase (not shown). Ethics Transgenic animals used for the experiments in this study were bred and used under the authority and ethical approval of the UK Home Office (Project Licence PPL60/3785). Results Rep8 interacts with p97 In a yeast two-hybrid screen of a HeLa cell cDNA library, using human p97 as a bait, the conserved but uncharacterized protein named, Rep8 or Ubxd6 (Swiss-Prot accession: O00124), was isolated (Fig. 1A). The human protein is 73% identical to its murine orthologue and 39% identical to its orthologue in zebrafish (Fig. S1). Database analyses indicate that Rep8 is only found in higher eukaryotes. The primary structure of Rep8 revealed that the protein contains a signal sequence followed by a transmem- brane domain, while the C-terminus contains a UBX domain (Fig. 1B and Fig. S1). The UBX domain is regarded as a general p97-interacting domain [24], indicating that Rep8 was a valid target of p97 in the yeast two-hybrid screen. In addition, the R..FPR motif known from other UBX proteins to play a critical role in p97 binding [22] is conserved in Rep8. By unbiased sequence searching, we also found that Rep8 shows some similarity to the transmembrane p97 cofactor known as SelS or VCP-interacting membrane protein (VIMP) (Fig. 1B and Fig. S1). The VIMP homology in Rep8 is located between the transmembrane region and the UBX domain and matches the major helical part of VIMP [PDB: 2Q2F]. In order to confirm the yeast two-hybrid interaction, GST, GST-tagged Rep8, and GST-tagged truncations of Rep8 (Fig. 1B) were expressed and purified from E. coli. To circumvent the likely folding issues of a recombinant transmembrane protein expressed in bacteria, we also deleted the first 56 amino acids encompassing the predicted signal sequence and transmembrane domain (Fig. 1B). The fusion proteins were used in precipitation experiments with p97, purified from human red blood cells. GST-Rep8 precipitated p97, whereas under the same conditions GST did not (Fig. 1C), thus confirming that Rep8 and p97 interact directly. Precipitation using the Rep8 truncations revealed that the C-terminal UBX domain was necessary and sufficient for p97 binding (Fig. 1C). To more thoroughly analyze the interaction between Rep8 and p97, we sought to map the Rep8 binding site on p97. Most p97- interacting proteins interact with either the N-domain on p97 or a short motif in the C-terminus. In general, UBX domain proteins interact with the N-domain [22]. Accordingly, precipitation experiments with GST-tagged Rep8 and various 6His-tagged p97 truncations (Fig. 2A) revealed that Rep8 interacts with the p97 N-domain (Fig. 2B). To analyze the interaction between endogenous p97 and Rep8, we raised an antibody to Rep8, and used it to probe p97 immunopre- cipitates for Rep8 from HeLa (Fig. 2C) and rat testis lysates (Fig. 2D). Again, we found that Rep8 was bound to p97 (Fig. 2C and 2D), showing that the two proteins are also associated in vivo. Rep8 is a transmembrane protein localized to the ER membrane Next, we sought to determine the subcellular localization of Rep8. To this end, the antibody to Rep8 was used to analyze PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25061