Protease K Protection Assay

Proteinase K protection assays were performed as described (21, 22). Briefly, equal aliquots of freshly prepared mitochondria were treated with varying concentrations of proteinase K (Thermo) for 20 min on ice. Triton X-100 (1%) was eventually added to permeabilize the mitochondrial membranes and allow proteinase K to reach the mitochondrial matrix. Samples were then subjected to SDS-PAGE and immunoblotting. TOM70/MFN1, TIM23/COX4, and TFAM were used to analyze proteinase K-mediated degradation of proteins located in the outer mitochondrial membrane (OMM), the inner mitochondrial membrane (IMM), the mitochondrial matrix (MM), respectively.