Par17 import into isolated mitochondria. A. Par17 and Par14 associate with mitochondrial surfaces in a salt-dependent manner. Yeast mitochondria were incubated with 35S-methionine labeled Par17 and Par14 lysates for 15 min at 25°C; Porin and GFP lysates were used as controls. Mitochondria were separated from soluble protein by the addition 500 mM sucrose buffer, subsequent centrifugation and additional washing in a 250 mM sucrose containing SEM buffer to ensure efficient separation of mitochondria from lysate ingredients. Mitochondria were then suspended in buffer containing increasing salt concentrations (0, 80 and 600 mM NaCl) and re-isolated by centrifugation. The samples within each panel including the respective reference sample (L, 20% for Porin and GFP, 50% for Par14 and Par17) were separated on the same gel to allow direct comparison (M +, mitochondria added). The amount of associated radioactively labeled protein was analyzed by SDS-PAGE and autoradiography. Mitochondrial association of Par14 and Par17 as displayed as bars on the diagram represent data normalized to the highest value including error bars (standard deviation for n = 3). B. Par17, but not Par14, is imported into isolated human, rat and yeast mitochondria. Left. Par17-QR and Par14 35S-methionine labeled lysates were incubated with different amounts of PK showing complete degradation of soluble parvulin proteins at a PK concentration of 50 μg/ml; at least 100 μg/ml PK were used for all following experiments. Right. Radiolabeled Par17-QR was incubated with mitochondria from human Jurkat cells, rat liver or from yeast either with or without subsequent proteinase K (PK) treatment (at least 100 μg/ml for 10 min). Mitochondria were then re-isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Par17 reached a PK protected compartment irrespective of the source of mitochondria indicating mitochondrial import. Par14, also present in this reaction, could be re-isolated together with mitochondria during centrifugation, but did not reach a protease-protected compartment. C. Time dependence of Par17 mitochondrial import. Mitochondria from yeast and human Jurkat cells were incubated with Par17-QR lysates for the times indicated followed by PK treatment. To assess relative import efficiencies, lysates of Su9-DHFR (amino acids 1–69 from subunit 9 of Neurospora crassa ATP synthetase fused to mouse DHFR ) were imported into the same mitochondrial preparations for equal time intervals at identical reaction conditions. Import reactions of Par17-QR and mature Su9-DHFR were analyzed by autoradiography and densitometric quantifications. Values were expressed as percent of added protein with correction for the different numbers of methionine residues in the precursor and processed proteins of Su9-DHFR. D. Par17 import depends on the mitochondrial membrane potential. Import reactions were performed as in B (right panel). Mitochondria were treated with the uncoupling reagent valinomycin to dissipate the mitochondrial membrane potential either before or after import reaction. Par17 was only imported into mitochondria with intact membrane potential (without valinomycin; or when the uncoupling agent was added after import). E. Par17 reaches the inner membrane of rat mitochondria. Porin and Par17-QR were imported into mitochondria isolated from rat liver. The outer mitochondrial membrane was removed by hypo-osmotic swelling as indicated by the disappearance of the porin signal. The Par17 band however only disappeared upon complete lysis of mitochondria by digitonin indicating that Par17 was imported at least to the inner mitochondrial membrane. F. Par17 is imported into the mitochondrial matrix. Import reactions with yeast mitochondria were performed with increasing amounts of digitonin leading to stepwise lysis. The amounts of Par17-QR, the inner membrane protein Tim23 and the matrix protein Mge1 were monitored in parallel samples using autoradiography for Par17 and Western blotting for Tim23 and Mge1. Tim23 is already degraded at a concentration of 0.1% digitonin, while Par17-QR and the matrix protein Mge1 resist PK. Degradation of these two proteins is only observed upon addition of 0.2% digitonin indicating that Par17 is transported to the mitochondrial matrix. Gels (upper part) and densitometric analysis (lower part) are shown.