Persistent PEX5 and PMP70 ubiquitylation in cells depleted of p97-UBXD8. (A) HEK293T cells were transfected with His-ubiquitin, PEX5-3xHA, and siRNAs to control, UBXD8, or p97. Cells were lysed in SDS, and denaturing HIS immunoprecipitations were performed. Immunoblots of indicated proteins showing increased ubiquitylation of PEX5 in cells depleted of UBXD8 and p97. N = 3 independent experiments. (B) HEK293T cells were transfected with HA-ubiquitin and siRNAs to control, UBXD8, or p97. Cells were lysed in SDS, and denaturing HA immunoprecipitations were performed. Immunoblots of indicated proteins showing increased ubiquitylation of PMP70 in cells depleted of UBXD8 and p97. N = 3 independent experiments. (C) Model showing p97-UBXD8 suppression of pexophagy. UBXD8 on peroxisomes recruits p97 to ubiquitylated peroxisome proteins such as PEX5 and PMP70. p97 ATPase activity extracts and degrades substrates to prevent their recognition by autophagy receptors. Source data are available for this figure: SourceData F10.