Figure 3. AtMic60 Is Localized in the Inner Membrane of Mitochondria with Its Soluble Domain Protruding in the IMS and Plays a Role in Mitochondria Morphology.

(A) Col0 callus grown in +Pi were subfractioned into mitochondria (see Experimental Procedures), microsomes (pellet fraction after centrifugation at 100,000 g of the supernatant obtained after the pellet of mitochondria at 16,000 g), and cytosol (supernatant fraction after centrifugation at 100,000 g of the supernatant obtained after the pellet of mitochondria at 16,000 g). Twenty micrograms of proteins from each fraction were used for western blots against marker proteins of each compartment.

(B) Mitochondria were ruptured and subfractioned into the membrane and soluble fraction. Twenty micrograms of proteins from each fraction were used for western blots against marker proteins of each fraction.

(C and D) Thermolysin treatments of purified mitochondria (C) or mitoplasts (mitochondria with ruptured OM; D) from Col0 callus grown in +Pi. One hundred micrograms of mitochondria or mitoplasts were incubated with 0–10 μg of thermolysin. Pellets were resuspended in 50 μl of SDS-PAGE buffer, and 10 μl was used for western blot analyses. Triton was used to completely disrupt membranes and to show that proteins are sensitive to protease when accessible.

(E) Col0 and the two atmic60 KO lines (Figures S3A and S3B) were stably transformed with a GFP-targeted mitochondrial construction [24]. Observations were done in leaves of 10-day-old seedlings grown in vitro. The scale bar represents 5 μm. Arrows indicated bigger mitochondria observed in atmic60 KO lines.

See also Figure S2.