Rep8 Is a Tissue-Specific p97 Cofactor Figure 3. Rep8 localizes to the ER membrane. (A) The insoluble fraction of a HeLa cells lysate was isolated by centrifugation. Pellets were mixed with sucrose, 0.5 M NaCl, 100 mM Na2CO3 or 0.1% SDS as indicated, and separated by centrifugation into a pellet (P) and supernatant fraction (S) prior to analysis by SDS-PAGE and blotting using antibodies to p97, calnexin, actin and Rep8. To ease comparison, the pellets were resuspended in the same volumes as the supernatant prior to analysis. All Rep8 was insoluble and only released with SDS, but not with sucrose, sodium chloride or sodium carbonate. (B) Confocal micrographs of formaldehyde-fixed HeLa cells transfected to express Rep8 with a C-terminal GFP-tag (left panel) and RFP modified to contain a signal sequence and an ER-retention signal (ER-targeted RFP; middle panel). In the merged image (right panel), the signals overlap (yellow). (C) Differential centrifugation of HeLa cells transfected to express Rep8-GFP as in (A). TMX3 and the proteasome subunit Rpn2 served as controls for a transmembrane and soluble protein, respectively. (D) Microsomes from HeLa cells expressing Rep8 with a C-terminal GFP tag were treated with proteinase K and Triton X-100 as indicated, before they were analyzed by SDS-PAGE and blotting. The C-terminus of Rep8 was detected by an antibody to the GFP-tag. The luminal ER protein, ERp57, served as a control. doi:10.1371/journal.pone.0025061.g003 immunofluoresence, HeLa cells were transiently transfected to express full length Rep8 with a C-terminal GFP-tag. The GFP-signal appeared at the ER, and co-localized with RFP that had been modified to contain a signal sequence and an ER retention signal (ER- targeted RFP) (Fig. 3B). Interaction with p97 was not required for the ER localization of Rep8, since a Rep8 truncation, lacking the UBX domain, also localized to the ER (not shown). Differential centrifugation revealed that, like endogenous Rep8, all the Rep8- GFP fusion protein was associated with the microsome pellet (Fig. 3C). To determine the membrane topology of Rep8, microsomes from transfected cells were treated with proteinase K. While the ER luminal protein ERp57 was resistant to proteinase K treatment (Fig. 3D), the Rep8-GFP signal disappeared completely upon treatment with proteinase K (Fig. 3D), indicating that the C- terminal GFP-tag is oriented towards the cytoplasm. As a control, the microsomes were treated with both detergent and proteinase K, which, as expected, led to degradation of both Rep8 and the luminal ERp57 (Fig. 3D). Rep8 is expressed primarily in reproductive tissue Previous studies have shown that Rep8 mRNA is primarily present in reproductive tissues [35]. To determine the tissue distribution of Rep8 on the protein level, we separated protein extracts from various rat tissues by SDS-PAGE and probed blots for the presence of Rep8. We found that Rep8 was almost exclusively expressed in testes and ovaries, but also present in the cell lines used here (Fig. 4A). Although both cell types used here are derived from human cancers, searching online databases (e.g. Oncomine, BioGPS) did not reveal any clear correlation between Rep8 expression and cancer (data not shown). Testes contain a mixture of different somatic cell types and germ cells at various stages of differentiation. In order to investigate which cell type in the testis is responsible for the high expression levels of Rep8, we performed qRT-PCR on developing testes from prepubertal mice undergoing the first wave of spermatogenesis. The most advanced male germ cells in prepubertal testes are undergoing mitotic proliferation at around 6 days post partum (dpp), progressing through the pachytene stage of meiosis at around 16 dpp, differentiating into early round spermatids at around 20 dpp, and starting to elongate at around 30 dpp. The Sdmg1 marker for somatic Sertoli cells [40], the Stra8 marker for mitotic (spermatogonia) and early meiotic (preleptotene spermatocyte) male germ cells [42], the Sycp3 marker for meiosis [43] and the Prm1 marker for round and PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e25061