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. 2015 Mar 18;290(18):11611–11622. doi: 10.1074/jbc.M115.642017

FIGURE 4.

FIGURE 4.

The temperature-sensitive mutant of mtHsp70, ssc1–42, can be inactivated by in vitro heat shock. A, WT and ssc1–42 cells were serially diluted and spotted on glucose (YPD)- or glycerol (YPG)-containing medium. Cells grew at the indicated temperatures. B, the indicated amounts of WT and ssc1–42 mitochondria (Mito) were analyzed by SDS-PAGE, Western blotting, and immunodetection with the indicated antisera. C, WT and ssc1–42 mitochondria were lysed with digitonin, and mitochondrial protein complexes were studied by blue native electrophoresis, Western blotting, and immunodetection with the indicated antisera. Cox, cytochrome c oxidase; Rip1, Rieske iron-sulfur protein; Atp, subunits of the F1FO-ATP synthase. D, 35S-labeled precursors of subunit 9 of the F1Fo-ATP-synthase fused to DHFR (Su9-DHFR, top panel), cytochrome b2(167)Δ fused to DHFR (center panel), and cytochrome c1 (Cyt1, bottom panel) were imported into WT and ssc1–42 mitochondria at the indicated time points. Where indicated, isolated mitochondria were subjected to in vitro heat shock prior to import. In control reactions, the membrane potential (Δψ) was dissipated prior to import. Proteins were detected by SDS-PAGE and autoradiography. p, precursor protein; m, mature protein.