Fig. 4: Rsp5 and Ubp16 control ubiquitylation of mitochondrial precursor proteins.

a, Normalized abundance–mass profiles of the TOM subunit Tom5, the E3 ubiquitin ligases Mdm30, Pep5 and Rsp5 (top), and the deubiquitylating enzymes Ubp16, Doa4 and Miy1 (bottom). The arrows depict profile peaks of co-migration of Tom5, Rsp5 and Upb16. b, WT and Tom20–His mitochondria (left) or WT and Tom40–HA cell extracts (right) were lysed with digitonin and affinity-purified through Ni-NTA agarose or anti-HA affinity matrix. Proteins were analysed using SDS–PAGE and immunodetection. Load, 0.5%; elution, 100%. The asterisk marks an unspecific signal of anti-Ubp16. c, Cell extracts of the indicated strains were analysed by immunodetection. p, precursor; m, mature form of Mdj1. Pre9, proteasomal subunit. d, WT, rsp5-1, mdm30∆ and mfb1∆ cells expressing cytochrome b₂-DHFR and His-tagged ubiquitin as indicated were lysed under denaturing conditions and affinity-purified through Ni-NTA agarose. Proteins were analysed using SDS–PAGE and immunodetection. Load, 0.2%; elution, 100%. e,f, WT and ubp16∆ (e) and WT, ubp16∆ and ubp16∆ rsp5-1 (f) strains expressing His-tagged ubiquitin were lysed under denaturing conditions and affinity-purified through Ni-NTA agarose. Proteins were analysed using SDS–PAGE and immunodetection. Load, 0.2%; elution, 100%. g, WT, ubp16∆, ubp16∆ rsp5-1 and ubp16∆pth2∆ cells expressing cytochrome b₂∆-DHFR and His-tagged ubiquitin as indicated were lysed under denaturing conditions and affinity-purified through Ni-NTA. Proteins were analysed using SDS–PAGE and immunodetection. Load, 0.2%; elution, 100%.