JCS024034 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Unlabeled Fis1 (TMC) molecules are not aggregated. Wild-type mitochondria were incubated with radiolabeled Fis1-TMC for 30 minutes at 25°C. Mitochondria were re-isolated by centrifugation and resuspended in labeling buffer containing, where indicated, IASD and TX-100. At the end of the labelling reaction all three samples were halved. One aliquot was re-isolated by centrifugation and solubilised directly in sample buffer whereas the other was resuspended in 0.1 M Na₂CO₃. After 30 minutes on ice, the samples were analyzed by flotation gradient centrifugation, where membranes float to the top of the gradient, and soluble aggregated proteins remain at the bottom. T, top fraction; M, middle fraction; B, bottom fraction. Tom20, a marker outer membrane protein; mHsp70, a soluble protein of the mitochondrial matrix.

• Supplemental Figure S2 -

  Fig. S2. The membrane insertion of Tom5 does not require the TOM complex. Radiolabeled precursor of Tom5 was incubated with intact mitochondria or mitochondria pretreated with either trypsin (A) or pSu9-DHFR (C). In B, D-F the import into mitochondria isolated from the indicated mutant strain was compared with that into mitochondria isolated from the corresponding wild-type strain. Modification with IASD was as described in the legend to Fig. 2. Mitochondrial proteins were separated by SDS-PAGE, blotted to a membrane and then analyzed by autoradiography.