FIGURE 3:

Tgl2 is imported into mitochondria via the Mia40 pathway. (A) Import of Tgl2 is dependent on Mia40. Mitochondria isolated from WT cells and cells depleted for Mia40 (Mia40↓) were incubated with radiolabeled Tgl2 for the indicated periods. After import, mitochondria were either treated with proteinase K (100 µg/ml) to digest nonimported Tgl2 (+PK) or left untreated (-PK). Next, mitochondria were reisolated and analyzed by SDS–PAGE and autoradiography. The autoradiography depicts a representative experiment; the quantification shows the mean of n = 3 independent experiments with SD bars. (B) Import of Tgl2 relies on redox-active sulfhydryl groups. Mitochondria isolated from WT cells were incubated with radiolabeled Tgl2 for the indicated periods. Tgl2 was preincubated with or without (+/−) NEM with or without (+/−) subsequent DTT treatment. All samples were treated with PK after import. (C) Tgl2 binds covalently to Mia40 as a mixed disulfide. After import of radioactively labeled Tgl2 for 10 min at 25°C, free thiols were blocked with NEM and mitochondria were lysed. Immunoprecipitation was performed with an antibody specific for Mia40 or preimmune serum. Bound proteins were eluted in either reducing (+DTT) or nonreducing (-DTT) buffer and analyzed by SDS–PAGE. The arrowheads depict Tgl2-containing protein bands. I, 10 or 20% of radiolabeled precursor protein used in each import reaction as indicated.