Fig. 5: The relationship of Pth2 to the quality-control factors Ubx2, Vms1 and Dsk2.

a, WT and Ubx2–HA mitochondria were lysed with digitonin and affinity-purified. Proteins were analysed using SDS–PAGE and immunodetection. Load 1%, elution 100%. b,c, WT, Tom40–HA and Tom40–HA pth2∆ (b) and Tom40–HA ubx2∆ (c) cell extracts were lysed with digitonin and affinity-purified. Proteins were analysed using SDS–PAGE and immunodetection. Load, 0.2%; elution, 100%. The asterisk marks an unspecific signal of anti-Ubp16. d, Serial dilutions of the indicated strains were spotted onto selective medium with glycerol as a carbon source and grown at 37 °C. EV, empty vector. e, Cartoon of the linear structure of Pth2. TM domain, transmembrane domain (amino acids 12–32). The amino acid exchange in the Pth2-mutant form is indicated. Cellular fractionation of the indicated strains. Post-nuclear supernatant (PNS) fractions enriched for mitochondria (P13) and the cytosol (S100) were analysed by immunodetection. Phosphoglycerate kinase 1 (Pgk1) was the cytosolic control. f, Serial dilutions of the indicated strains were spotted onto full medium with either glucose or glycerol as a carbon source. g, Cell extracts of the indicated strains were analysed by immunodetection. p, precursor; m, mature form of Mdj1. h, The proposed model of constitutive quality control for the removal of precursor proteins accumulated at the mitochondrial protein entry gate. Rsp5 ubiquitylates precursor proteins accumulated at the TOM complex. Ubp16 removes faulty ubiquitins from precursor proteins to enable their transport into the mitochondria. Ubx2 interacts with the TOM complex and cooperates with the AAA-ATPase Cdc48 in the transfer of ubiquitylated precursor proteins to the proteasome¹⁷. Pth2 binds to the TOM complex independently of Ubx2 and cooperates with Dsk2 in the transfer of precursor proteins to the proteasome. OM, outer membrane.