Reconstitution of Mitochondria Derived Vesicle Formation Demonstrates Selective Enrichment of Oxidized Cargo
Figure 2
Mitochondrial stress triggers MDVs in vitro.
A) Isolated mitochondria were incubated in the absence of cytosol, with an energy regenerating system, for 60 minutes at 37°C (or 4°C) under the indicated conditions: 10 µM oligomycin, 50 µM Antimycin A, 50 µM Chloramphenical, 0.1 mM ATPγS, 200 µM xanthine and 0.4U xanthine oxidase. Along with 2 µg of the starting mitochondria, supernatants of the reactions were separated by SDS-PAGE for analysis by western blot, probing for anti-Core 2, a component of mitochondrial electron transport chain complex III, and the outer membrane channel VDAC. At the exposures shown here, the VDAC western blot does not detect the signal within the starting mitochondria. The data is representative of at least 3 independent experiments. B) As in A) except that 20 mM of NEM was added when indicated, and 0.1 mM GTPγS was used where indicated. C) Supernatants obtained from reactions performed in the presence of either 200 µM xanthine and 0.4U xanthine oxidase or 50 µM Antimycin A were fixed, and isolated in low-melt agarose and prepared for EM analysis. Images show thin sections from the pelleted MDVs, revealing both single and double-membrane bound vesicles. Note the differential electron electron density of material within the inner membrane compared to the space between the two membranes. Scale bars are all 100 nm.