Louise Kjær Klausen:
Identification and Characterization of the Novel p97 co-factors, Rep8 and ASPL

Date: 01-12-2012    Supervisor: Rasmus Hartmann-Petersen

The highly conserved and ubiquitin-specific AAA ATPase p97 acts on ubiquitylated substrates in diverse cellular mechanisms such as chromatin-associated degradation, fusion of homotypic membranes and ER-associated degradation. Different p97 cofactors associate with the ATPase, thereby constituting different complexes that guide the ATPase to its many cellular functions. In this study, two novel p97 cofactors, the human Rep8 and ASPL proteins, were identified and characterized. ASPL was found to rescue the cycloheximide sensitive phenotype of the budding yeast ubx4Δ mutant, and ASPL must therefore be a functional orthologue of the yeast Ubx4. Unlike Ubx4, ASPL is not involved in the degradation of ERAD substrates. ASPL was found to bind p97 in vivo and in vitro, and it requires a central region containing both a SHP box and a UBX domain for binding to the N-domain of p97. Localization studies showed that ASPL localizes to the ER membrane and in vitro ASPL leads to disassembly of the p97 hexameric ATPase. Rep8 was found to interact with p97 both in vitro and in vivo, and the binding was mediated through the N-domain of p97 and the UBX domain of Rep8. Localization studies showed that Rep8 localizes to the ER membrane with the UBX domain situated in the cytosol. Mouse Rep8 is highly tissue-specific and abundant in gonads. In tests, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Additional precipitation experiments revealed that Rep8 associates in vivo with the ERAD component, Hrd1, and knock-down of Rep8 expression in human cells leads to a retarded degradation of an ERAD substrate. In addition, Rep8 knock-down decreased the association of p97 with the ER membrane. Thus, Rep8 is a gonad specific protein that tethers p97 to the ER membrane for efficient ER-associated degradation.