Fig. 2. MTP18, a putative internal mitochondrial TM protein. (A) Hydrophobicity plot for MTP18 as measured by the TMpred algorithm showing probability for TM domains. (B) Western blot of mitochondrial extracts treated with Na₂CO₃ and NaCl after separation by ultracentrifugation in soluble (S, peripheral membrane proteins) and pelleted proteins (P, integral membrane proteins). Blots were probed with anti-Hsp60, anti-hFis1 and anti-MTP18. (C) Protease digestion of mitochondria with proteinase K (PK) or trypsin (lanes 2 and 4, or 3, respectively). Extracts were subjected to SDS-PAGE and western blots were carried out with anti-MTP18, anti-Mfn1, anti-cytochrome c and anti-Hsp60. Mitochondria were also osmotically shocked to disrupt their outer membrane and treated with PK (lane 4). MTP18 becomes degraded like cytochrome c upon osmotic shock. (D) Confocal microscopy of mitochondria from MTP18-myc transfected COS-7 cells stained with anti-MTP18 (green) and anti-Hsp60 (red). MTP18-myc did not completely colocalise with the mitochondrial matrix protein Hsp60. Arrows indicate arcas of mitochondria where MTP18 does not colocalise with Hsp60; double arrow indicates mitochondria from an untransfected cell.