FIG. 4.

PNPase exists in a multimeric IMS complex and is mobilized with tBID treatment. (A) Mouse liver mitochondria were size fractionated by BN-PAGE and immunoblotted for PNPase or respiratory complex IV. (B) PNPase is partially mobilized from mitochondria. Isolated mouse liver mitochondria were treated with or without tBID for 15, 30, 60, and 120 min and subjected to centrifugation. Supernatants (s) and mitochondrial pellets (p) were harvested and analyzed by immunoblotting. PNPase, cytochrome c, and Timm13 (data not shown) were released from mitochondria. Data are representative of three independent experiments. (C) Radiolabeled TCL1 was not imported into isolated yeast mitochondria at 25°C in the presence or absence of Δψ. After import, samples were treated with trypsin to remove nonimported precursor, and treatment was stopped with soybean trypsin inhibitor. Import-competent controls were matrix-localized Su9-DHFR and IMS-localized cytochrome c₁. Samples were analyzed by SDS-PAGE and fluorography. Standard (Std) refers to 10% of the radioactive precursor added to each assay. p, precursor; i, intermediate; m, mature.