Figure 4(A) Total cell lysate (T), post-mitochondrial supernatant (PMS), and HistodenzTM-purified mitochondria (M+P, M) from the indicated yeast strains (wild type, pex19Δ, and pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for PEX13-V5 (α-V5) and porin (α-porin). (B) Digitonin-solubilized (1 or 4 g/g digitonin/protein ratio, as indicated) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by BN-PAGE with a 3-18% gradient and stained with Coomassie brilliant blue. BHM, bovine heart mitochondria; V2, complex V-dimer; SL,supercomplex III2IV2; Ss, supercomplex III2IV1; V1, complex V-monomer. (C) Digitonin-solubilized (1 or 4 g/g digitonin/protein ratio, as indicated) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by BN-PAGE with a 3-18% gradient and immunoblotted for PEX13-V5 (α-V5). Green box for wild type, orange box for pex19Δ, purple box for pex19Δmsp1Δ, indicates gel area of 300-130kDa correlated to complexome profiling heatmap. (D) Heatmap representing the protein abundance of Pex11, 13, 14, 17, 25 in digitonin-solubilized (3 g/g digitonin/protein ratio) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor). Green box for wild type, orange box for pex19Δ, purple box for pex19Δmsp1Δ, indicates gel area of 300-130kDa correlated to complexome profiling heatmap. (E) Heatmap representing the protein abundance of Mdh3 and Pot1 in digitonin-solubilized (3 g/g digitonin/protein ratio) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor). (F) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pex22-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Pex22-V5 (α-Pex22-V5) and porin (α-porin). (G) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ ) expressing Pot1-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pot1-V5 (α-Pot1-V5) and porin (α-porin). (H) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1 Δ, and pex13Δmsp1Δpex19Δ) expressing Mdh3-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Mdh3-V5 (α-Mdh3-V5) and porin (α-porin). (I) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pot1-V5 (under its endogenous promotor) were either left untreated (nt) (lane 1,4,7) or were treated with proteinase K in high fidelity buffer (PK-HF) (cutting control, lane 2,5,8) or low fidelity buffer (PK-LF) (protease protection assay, lane 3,6,9) and separated by SDS-PAGE and immunoblotted for Mia40 (intermembrane space) (α-Mia40), Pot1-V5 (α-V5), porin (outer membrane) (α-porin) and Tom22 (outer membrane but partially proteinase exposed) (α-Tom22). (J) Cartoon representing the predicted topology of the peroxisomal importomer assembled on mitochondria.