Tnt rabbit reticulocyte lysate

Radiolabeled ([³⁵S]l-methionine) TbTim17 and TbAAC were synthesized using a cell-free transcription-and-translation (TNT) rabbit reticulocyte lysate (Promega). Isolated T. brucei mitochondria (100 µg) were suspended in 90 µl of import buffer [250 mM sucrose, 80 mM potassium chloride, 5 mM magnesium chloride, 5 mM dithiothreitol, 1.0 mg/ml fatty acid-free bovine serum albumin, 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS)–KOH (pH 7.2), 2 mM ATP, 10 mM creatine phosphate, 0.1 mg/ml creatine kinase, 8 mM potassium ascorbate, 200 nM N,N,N′,N’-tetramethylphenylenediamine, 5 mM nicotinamide adenine dinucleotide (NADH)]. The mitochondrial suspension was mixed with 10 µl of radiolabeled precursor protein TNT lysate and incubated at 27°C for up to 15 min. Some mitochondria were pretreated with 50 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 15 min to deplete mitochondrial membrane potential (−Ψ) before the addition of radiolabeled precursor proteins. Mitochondria were washed twice with SEM buffer to remove excess radiolabeled protein. For TbTim17 and TbAAC protein imports, mitochondria were resuspended in SEM buffer (1 µg/µl) and were treated with PK (20 µg/ml) for 10 min on ice. Postimport fractions were analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by developing exposed autoradiography films.