|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. The insertion of Fis1 does not require TOM import receptors. (A) Radiolabeled precursor of Fis1-TMC was incubated with intact mitochondria or mitochondria pretreated with trypsin. Modification with IASD was as described above. Mitochondrial proteins were separated by SDS-PAGE, blotted to a membrane and then analyzed by autoradiography and immunocytochemistry. The antibodies used were directed against the receptor proteins Tom70 and Tom20 and the membrane-embedded Tom40, where a small fragment is cleaved off upon trypsin treatment. Lower panel: as a control, radiolabeled porin was incubated with the intact or trypsin-pretreated mitochondria for the indicated time periods. Mitochondria were treated with proteinase K (100 µg/ml) to degrade non-inserted protein and mitochondrial proteins were analyzed by SDS-PAGE and autoradiography. Insertion of Fis1 was calculated as the fraction of bound material (–IASD), which is protected in the presence of IASD (lower band in +IASD). The amount of precursor imported into intact mitochondria after 20 minutes was set to 100%. (B) Radiolabeled Fis1-TMC was incubated with mitochondria isolated from either the wild type or from cells lacking Tom20. Lower panel: as a control, radiolabeled porin was incubated with the same mitochondria and further treatment and analysis was as described in A. (C) Radiolabeled Fis1-TMC was incubated with mitochondria isolated from either the wild type or from cells lacking Tom70. Lower panel: as a control, radiolabeled porin was incubated with the same mitochondria and further treatment and analysis was as described in A. (D) Radiolabeled Fis1-TMC was incubated at 0°C for the indicated time points with either wild-type mitochondria or with mitochondria isolated from cells lacking either Tom20 or Tom70. Further treatment was as described in the legend to Fig. 2D. The bands were quantified and the intensity of the band corresponding to the unmodified protein was taken as a measure for protein insertion. The amount of protein inserted into mitochondria isolated from the corresponding wild-type strain after initial incubation for 20 minutes was set at 100%.
|
