Rep8 Is a Tissue-Specific p97 Cofactor Figure 4. Expression of Rep8. (A) The indicated rat tissues and cell extracts were analyzed by SDS-PAGE and blotting using antibodies specific for Rep8 (upper panel) and tubulin (lower panel). Rep8 was expressed almost exclusively in testes, ovaries and in the cell types used in this study. (B) qRT- PCR for Rep8 expression during mouse testis development. Bars indicate mean expression relative to b-actin, normalized to the maximum expression level for that gene during the developmental time course. Error bars indicate standard errors. (C) qRT-PCR for Rep8 expression in adult Tex19.12/2 testes. Mean expression levels relative to b-actin were normalized to control adult testes for each gene. Error bars indicate standard errors. (D) In situ hybridization of Rep8 to adult mouse testes. Bound sense or anti-sense Rep8 probes were visualized with dark blue/purple precipitate. Sections were counterstained with nuclear fast red. Scale bar 100 mm. (E) Higher magnification images of Rep8 in situ hybridization to adult testes. The approximate seminiferous epithelial stage is indicated by roman numerals, and examples of mitotic spermatogonia (sg), meiotic spermatocytes (sc), round spermatids (rs) and elongated spermatids (es) are annotated. Scale bar 20 mm. (F) In situ hybridization of Rep8 to adult mouse ovary. Scale bar 100 mm. Granulosa cells (gr), oocytes (oo) and corpora lutea (cl) are indicated. doi:10.1371/journal.pone.0025061.g004 elongating spermatids [44] were all expressed, as would be expected during this developmental time course (Fig. 4B). Rep8 expression in prepubertal testes increased dramatically between the 16 dpp and 29 dpp time points, coincident with the appearance of late meiotic spermatocytes and post-meiotic round spermatids in the testis (Fig. 4B). Furthermore, adult testes from Tex19.12/2 mice, which contain all the somatic cell types in the testis, but have significantly reduced numbers of late meiotic spermatocytes and post-meiotic spermatids [41], have reduced Rep8 expression (Fig. 4C). Thus, the high levels of Rep8 expression in the testis depends on the presence of late meiotic spermatocytes/post-meiotic round spermatids in this tissue. In order to test whether Rep8 is expressed in the developing germ cells in adult testes, we performed in situ hybridization. In adult mouse testes, high Rep8 expression was evident in the developing germ cells within the seminiferous tubules (Fig. 4D). Rep8 mRNA levels are low or undetectable in the interstitial somatic cells in the testis (Fig. 4D), in mitotic or meiotic male germ cells inside the testis tubules (Fig. 4E), and in early stages of post- meiotic round spermatid differentiation (Fig. 4E, stage I–VI). Rep8 mRNA is abundant during the late stages of round spermatid differentiation (Fig. 4E, stage VII–VIII), and as the round spermatids start to elongate (Fig. 4E, stage IX–X). However, Rep8 mRNA levels are low or undetectable in elongated spermatids (Fig. 4E, stage I–VI, stage VII–VIII). These in situ hybridization data are consistent with the qRT-PCR data for Rep8 expression in the testis. Thus, the high levels of Rep8 expression in the testis appear to be caused primarily by a pulse of Rep8 upregulation in germ cells during late round spermatid differentiation. In order to test whether the high levels of Rep8 mRNA expression in the adult ovary [35] are also caused by expression in the developing germ cells, we performed in situ hybridization on adult ovaries. In contrast to the testis, the high levels of Rep8 expression in the ovary appear to be caused primarily by expression in the somatic cells rather than the germ cells (Fig. 4F). Rep8 mRNA levels are low or undetectable in the oocytes, and in the somatic corpora lutea and stromal cells present in the ovary (Fig. 4F). However, Rep8 mRNA is abundant in the somatic granulosa cells that surround the oocyte in the developing follicles (Fig. 4F). The high levels of Rep8 expression in ovaries and testes therefore does not appear to be caused by a common Rep8- expressing cell lineage in male and female gonads. Rather, Rep8 expression in adult gonads may be a consequence of some similarity between the cell biology of granulosa cells in the ovary and round spermatids in the testis. Rep8 associates with Hrd1 Since we found that Rep8 is a cofactor of p97 that localizes to the ER membrane, we analyzed if Rep8 was associated with the E3 ubiquitin-protein ligase, Hrd1, that plays an important role in ERAD [8]. Extracts from HeLa cells, stably transfected to express biotin-tagged Hrd1, were precipitated with a streptavidin resin. Indeed, the biotinylated Hrd1 interacted with Rep8 (Fig. 5A). However, since we could not detect any interaction between recombinant Hrd1 and Rep8 purified from E. coli (not shown), the interaction observed in HeLa cells is probably indirect and bridged by p97 or other components of the ERAD system. Knock-down of Rep8 expression inhibits ERAD and p97- membrane interaction Since Rep8 is an ER-membrane protein that interacts with p97, it is possible that proteolysis of ER proteins is impaired in cells with a decreased content of Rep8. To test this prediction, Rep8 expression in MelJuSo cells was knocked down with siRNA (Fig. 5B). Knock-down of Rep8 did not alter the level of p97 (Fig. 5B). Thus, cells do not compensate for the lack of Rep8 by inducing p97. To test if Rep8 plays a role in ERAD, the degradation kinetics of the model ERAD substrates, TCRa and CD3d, were analyzed. In both cases we observed a slight retardation in their degradation (Fig. 5C and Fig. S2). However, the degradation of a cytoplasmic p97-relevant proteasome substrate was unaffected (Fig. S3), indicating that Rep8 specifically targets ER-derived proteasome substrates. We then speculated if Rep8 perhaps recruits p97 to the ER membrane. By differential centrifugation and quantitative immu- noblotting, the amount of p97 associated with the microsome pellet was determined. In cells transfected with Rep8-specific siRNA, the amount of membrane-bound p97 was significantly reduced compared with the control (Fig. 5D). We therefore conclude that Rep8 tethers p97 to the ER membrane. Discussion The ATPase complex, p97, is a molecular segregase [17] connected with a broad spectrum of cellular pathways, including fusion of ER and Golgi membranes [19,45], DNA repair [46], transcription factor activation [18] and ERAD [11,47]. The cellular mechanisms involved in directing p97 to these various functions are probably determined by its range of cofactors. Here, a previously cloned gene [35], encoding the uncharacterized human protein Rep8, was isolated as a p97 cofactor. We found that Rep8 expression is highly tissue specific and most abundant in gonads. This is in accordance with previous results on the mRNA level [35]. Furthermore, we have shown that the high levels of Rep8 expression in the testis is caused by expression in the post-meiotic round spermatids within this tissue. Rep8 is not the only ERAD relevant protein known to reside in the testes. Previous studies have found testis-specific homologues of calnexin (Clgn) and protein disulfide isomerase (Pdilt) interact to form a testis- specific chaperone complex in post-meiotic germ cells [48–50]. Sperm from Clgn2/2 mice are infertile and exhibit defects in the heterodimerization and maturation of egg-binding glycoproteins in the ER during spermatogenesis. These egg-binding proteins are not present on the surface of mature Clgn2/2 sperm, and Clgn2/2 PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25061