For the preparation of mitochondria, third-instar larvae or flies were homogenized in ice-cold isolation buffer STE+BSA (250 mM sucrose, 5 mM Tris, 2 mM EGTA, 1% (w/v) BSA, pH 7.4) using a 15 ml Dounce homogenizer. Cellular debris were pelleted at 1000 g for 5 min and supernatants were transferred to new tubes. Mitochondria were washed two times and final mitochondrial pellets were resuspend in 1 ml STE buffer in the presence of 200 µg/ml emetine (Sigma) and 100 µg/ml cycloheximide (Sigma) to inhibit cytoplasmic translation. Protein concentrations were determined using the Bradford assay.
In organello transcription assays were performed as described [45] (link) using 200 µg mitochondria/sample and a modified transcription buffer (25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 10 mM K2HPO4, 50 µM EDTA, 5 mM MgCl2, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris-HCl (pH 7.4) and 1% (w/v) BSA). In short, after labeling, mitochondrial RNA was isolated using Totally RNA kit (Ambion). Mitochondrial RNA was fractionated on 1.2% agarose gels and blotted to Hybond-N+ membranes (Amersham Biosciences).
In vitro assays to study mitochondrial de novo translation with [35S]-methionine were performed as described [46] (link). Equal amounts of total mitochondrial protein were loaded on 15% SDS-PAGE gels. Gels were fixed in isopropanol-acetic solution, stained with Coomassie, destained in ethanol-acetic acid solution and treated with Amplify Solution (GE Healthcare). Afterwards gels were dried and [35S]–methionine-labelled proteins were visualized by autoradiography. The mitochondrial translation profile was compared to previously published profiles in Schneider cell lines [47] (link), additionally ND2 and ATP6 were identified by endopeptidase fingerprinting in the second dimension (data not shown) [48] (link).
In organello transcription assays were performed as described [45] (link) using 200 µg mitochondria/sample and a modified transcription buffer (25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 10 mM K2HPO4, 50 µM EDTA, 5 mM MgCl2, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris-HCl (pH 7.4) and 1% (w/v) BSA). In short, after labeling, mitochondrial RNA was isolated using Totally RNA kit (Ambion). Mitochondrial RNA was fractionated on 1.2% agarose gels and blotted to Hybond-N+ membranes (Amersham Biosciences).
In vitro assays to study mitochondrial de novo translation with [35S]-methionine were performed as described [46] (link). Equal amounts of total mitochondrial protein were loaded on 15% SDS-PAGE gels. Gels were fixed in isopropanol-acetic solution, stained with Coomassie, destained in ethanol-acetic acid solution and treated with Amplify Solution (GE Healthcare). Afterwards gels were dried and [35S]–methionine-labelled proteins were visualized by autoradiography. The mitochondrial translation profile was compared to previously published profiles in Schneider cell lines [47] (link), additionally ND2 and ATP6 were identified by endopeptidase fingerprinting in the second dimension (data not shown) [48] (link).
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