FIG. 4.

Coa2 is a soluble matrix protein loosely associated with the IM. (A) Immunoblot of mitochondria (mito) and the postmitochondrial fraction (PMF) purified from the COA2-3HA strain. Pgk1 is a cytosolic marker, and Por1 is a mitochondrial outer membrane protein. (B) Purified mitochondria from the COA2-3HA strain were sonicated in 20 mM HEPES, pH 7.4, and the indicated concentration of NaCl. The soluble and insoluble fractions were separated by centrifugation at 100,000 × g for 1 h and analyzed by immunoblotting. Cyt1 is an integral IM protein, Cyc1 is associated with the IM, and Sod2 is a matrix soluble protein. (C) Purified mitochondria from the COA2-3HA strain were incubated in 20 mM HEPES, pH 7.4, with 1.2 M sorbitol (intact) or without it (hypotonic), for 30 min on ice, digested on ice for 40 min with the indicated concentration of proteinase K (PK) (μg/ml), and analyzed by immunoblotting. (D) Purified mitochondria from the COA2-3HA strain were sonicated or incubated in 20 mM HEPES, pH 7.4, with 1.2 M sorbitol (intact) or without it (hypotonic), for 30 min on ice. The soluble (S) and insoluble (P) fractions were separated by centrifugation at 15,000 × g for 20 min and analyzed by immunoblotting. Ccs1 is a soluble IMS protein. (E) Purified mitochondria as described in panel C were either sonicated (+) three times with 30-s pulses to rupture them or left intact (−). The samples were then treated with 100 μg/ml proteinase K (PK) at 24°C. At each time point (5, 10, and 20 min), 10 μg of total protein was removed for immunoblotting.