Figure 2.

hSOD1 resides in the mitochondrial matrix. A, hSOD1 is associated with mitoplasts in a high salt-resistant manner. Mitochondria were subfractionated into MPs and PMSs, and MPs were subsequently incubated in the presence of 150 mm KCl. After centrifugation, MP and PMS fractions were immunoblotted for SOD1, the intermembrane component cytochrome, as well as the matrix component MnSOD. Cyt. c, Cytochrome c. B, Disruption of the mitochondrial IM is required to immunoprecipitate hSOD1 associated with mitoplasts. Intact or sonicated MPs were incubated with SOD1 antibody (SOD1 Ab)-protein G-Sepharose and then centrifuged at 600 × g for 30 s. Samples were immunoprobed for SOD1. MnSOD and COX I antibodies were used as markers of a soluble and an IM component of the mitoplasts, respectively. Lanes 6 and 7 represent a negative control without the addition of SOD1 antibody. I, Mitoplast input; S, supernatant; P, immunoprecipitated pellet. C, Swelled mitochondria from G85R brain were incubated in the absence or presence of 20 μg/ml PK (Prot. K) for 20 min on ice, with and without the addition of 10 mm CHAPS (lanes 2, 3) or with and without sonication (lanes 4, 5). The mouse SOD1 (mSOD1) was resistant to proteolysis in contrast to the human G85R-SOD1. Hsp60, a matrix component, and the mitochondrial IM protein COX I were used as markers to assess IM integrity. Lane 1 contains swelled mitochondria without treatment with PK, detergents, or sonication. D, Liver mitochondria from a 10-month-old G85R mouse treated as in C.