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. 2007 Nov 5;179(3):389–395. doi: 10.1083/jcb.200707123

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Copyright © 2007, The Rockefeller University Press

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Figure 4. — Mia40-dependent protein import is influenced by the activity of the respiratory chain. (A) Radiolabeled Cox19 protein was incubated with wild-type, Δrip1, and Δcox18 mitochondria in the presence of different concentrations of DTT. Nonimported protein was removed by treatment with proteinase K on ice. Mitochondria were reisolated and dissolved in sample buffer. Proteins were analyzed by SDS-PAGE and autoradiography. Imported proteins were quantified by densitometry. Import efficiencies without DTT were set to 100% (control). (B) Radiolabeled Tim10 was imported into mitochondria from a wild-type and a Δcyc1/Δcyc7 mutant as described in A. (C) Wild-type mitochondria were incubated in the absence or presence of 100 μg/ml antimycin A and 10 mM potassium cyanide for 3 min at 25°C before radiolabeled Tim10 was imported. Mitochondria were treated with 65 mM iodoacetamide and reisolated, and proteins were analyzed by nonreducing SDS-PAGE. This allows the identification of monomeric Tim10 as well as of Mia40-associated Tim10 (Mia40 • Tim10).