Rep8 Is a Tissue-Specific p97 Cofactor Figure 5. Rep8 binds Hrd1 and participates in ERAD. (A) HeLa cells expressing in vivo biotinylated Hrd1 were precipitated using streptavidin Sepharose beads, or as a control, IgG Sepharose beads, and analyzed by SDS-PAGE and blotting using the indicated probes. Rep8 was bound to the immobilized Hrd1. (B) Human MelJuSo cells were transfected with control siRNA or two different siRNAs specific for Rep8. After 5 days, the expression of the indicated proteins was analyzed by SDS-PAGE and blotting. Actin served as a loading control. (C) Pulse-chase experiments performed on cells expressing YFP-tagged CD3d. The cells were transfected with Rep8 siRNA#2 (filled square) or control siRNA (filled circle). At the indicated times during the chase period the substrate was retrieved by precipitation using antibodies specific for GFP. The precipitated material was resolved by SDS- PAGE and visualized by phosphoimaging (top panel). Knock-down of Rep8 expression caused a decrease in the degradation rate (lower panel). (D) The amount of membrane-bound p97 in MelJuSo cells transfected with control siRNA or siRNA specific for Rep8 was determined by differential centrifugation and quantitative blotting. The error bars show the standard deviation. The difference was significant at the p,0.01 level (**) (t-test, n = 4). doi:10.1371/journal.pone.0025061.g005 sperm have defects in egg-binding at fertilization [51–53]. Interestingly, like Rep8, Clgn and Pdilt are highly expressed in round spermatids, a stage of spermatogenesis where cell-surface and secreted proteins, that will eventually facilitate interactions between the sperm and the egg during fertilization, are being synthesized. Perhaps the special pH, temperature and redox environment, or the presence of specific substrates in round spermatids may require expression of ERAD proteins such as Rep8. Indeed, components of the insoluble protein matrix that is present in the specialized acrosome structure, where many of these secreted proteins are stored, may be prone to aggregation and misfolding [54]. The generation of Rep82/2 knockout mice will be required to analyze the role of Rep8 in reproductive tissues in more detail. Although nematodes do not encode any apparent orthologues of Rep8, simultaneous knock-down of several UBX domain proteins results in a germ-line phenotype that does not produce sperm [55]. Perhaps mice lacking Rep8 will display a similar phenotype. Rep8 contains a transmembrane domain, a VIMP-like region and a UBX domain. VIMP is also a transmembrane protein that localizes to the ER-membrane and plays a role in ERAD [29]. It is known that VIMP associates with p97 [29], but VIMP does not contain a UBX domain and the p97 interaction region in VIMP has yet to be identified and characterized. Another important difference between VIMP and Rep8 is that VIMP is most likely a selenoprotein whereas Rep8 is not. The region in VIMP which is homologous to Rep8 is located just next to the transmembrane domain. The structure of this region of VIMP is basically an extended helix [PDB: 2QF2], which probably functions as a spacer to keep the VIMP C-terminal area at a distance from the membrane. Presumably the VIMP-like area in Rep8 shares this function. The UBX domain of Rep8 is sufficient and necessary for interacting with the p97 N-domain. We also found that Rep8 interacts with the E3 ubiquitin-protein ligase Hrd1. However, since we were unable to reconstitute this interaction using purified components in vitro, we speculate that this interaction is indirect and bridged by other components of the ERAD machinery, perhaps by p97 itself, which interacts with Hrd1 directly [56–58]. Since Rep8 and Hrd1 both associate with the p97 N-domain their interaction with p97 is probably mutually exclusive. However, due to the hexameric structure of p97, the ATPase may in principle associate simultaneously with up to six different N-domain binding partners, and ternary Rep8-p97-Hrd1 complexes could therefore be relevant. For some p97 cofactors, interaction critically depends on p97’s prior association with other cofactors [59]. In the future it would be interesting to see if Rep8 and VIMP co-associate with p97. PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25061