FIG. 2.

Separation of mtHsp70 into domains and import into isolated mitochondria. (A) Ssc1 constructs used. (B) Import into mitochondria. Rabbit reticulocyte lysates with radiolabeled mitochondrial preproteins (2% [vol/vol] of import reaction mixture) were incubated with isolated yeast wild-type mitochondria (25 μg of mitochondrial protein/100 μl of import reaction mixture) for the indicated times in the presence (+) or absence (−) of a ΔΨ. Import was stopped by the addition of 1 μM valinomycin. Half of the samples were treated with proteinase K (Prot. K). After reisolation of the mitochondria and separation by SDS-PAGE, imported proteins were detected by digital autoradiography. The asterisk indicates a fragment of Ssc1(AP) that was generated in small amounts by proteinase K upon incubation of the construct with mitochondria in the presence of a ΔΨ, apparently representing an incompletely imported membrane-spanning form of the protein; the quantitations shown in the following figures did not include this fragment but are only based on the fully imported mature forms. m, mature form; p, precursor form.