RNF5 Regulates HERP1 and OS9 Stability and Modulates Processing of an ERAD Substrate

Abstract

Endoplasmic reticulum-associated degradation (ERAD) must dynamically adapt to cellular protein-folding demands, yet mechanisms regulating ERAD-associated adaptors remain incompletely defined. Here, we identify the E3 ubiquitin ligase RNF5 as a regulator of the ERAD adaptors HERPUD1 (HERP1) and OS9. RNF5 depletion increased HERP1 and OS9 protein abundance, while semiquantitative endpoint RT-PCR did not reveal obvious changes in their transcript signals. Available cycloheximide-chase experiments were consistent with increased adaptor persistence and more rapid loss of the ERAD-L substrate null Hong Kong α1-antitrypsin (NHK) after RNF5 depletion. In contrast, expression of the catalytically inactive RNF5 C42S mutant was associated with slower NHK loss, indicating that RNF5 depletion and catalytic inactivation produce distinct functional outcomes. Proteomic screening and co-immunoprecipitation supported association of RNF5 with HERP1- and OS9-containing complexes. RNF5 depletion reduced compound 225-induced HERP1 ubiquitination and adaptor loss, while wild-type RNF5 produced stronger HERP1 ubiquitination than C42S, supporting a contribution of RNF5 catalytic activity. RNF5 depletion also attenuated selected tunicamycin-induced UPR markers and 225-associated cytotoxic/apoptosis-related readouts while increasing SA-β-galactosidase- and γH2AX-associated phenotypes. Together, these findings identify RNF5 as a regulator of ERAD-adaptor abundance and NHK processing, while leaving the causal relationship between adaptor regulation and the RNF5-depletion phenotype to be resolved by formal rescue and epistasis studies.