PINK1-Interacting Proteins: Proteomic Analysis of Overexpressed PINK1
Figure 3
Cellular abundance of potential mitochondrial PINK1-interacting proteins. Mitochondrial and cytosolic fractions from fibroblasts were analyzed by Western blotting using antibodies against HSP60, LRPPRC, TUFM, MT-CO1, and GRP75. (a) The mitochondrial localization of LRPPRC and TUFM was confirmed, and no differences in their cellular abundance were detected when comparing PINK1 mutants and controls. Furthermore, the level of LRPPRC-associated MT-CO1 was not altered in PINK1 mutants. (b) In the mitochondrial fractions, the abundance of (processed) GRP75 was comparable in PINK1 mutants and controls under basal and valinomycin stress conditions (1 μM for 24 h). In the cytosol, an additional band representative of accumulation of nonprocessed MTS-GRP75 was detected when cells were treated with the mitochondrial membrane inhibitor valinomycin. Due to a possible contamination of the cytosolic fraction with mitochondria and/or partially cytosolic localization of GRP75, also the processed form of the protein is apparent in this fraction. The mitochondrial marker VDAC1 and the cytosolic marker β-actin served as loading controls. GRP75: 75 kDa glucose-regulated protein; HSP60: Heat shock 60 kDa protein; LRPPRC: Leucine-rich PPR motif-containing protein; MT-CO1: Mitochondrially encoded cytochrome c oxidase I; MTS-GRP75: GRP75 with mitochondrial targeting sequence; TUFM: Elongation factor Tu; VDAC1: Voltage-dependent anion channel 1.
