Figure 3. N+C cells have a strongly impaired import via the TIM23 complex.
(A) Total cell extracts of FL and N+C cells grown at 24°C and 30°C were analyzed by SDS–PAGE and immunoblotting using indicated antibodies. p - precursor, and m - mature form of Mdj1. (B–G and I–J) 35S-labeled mitochondrial precursor proteins were imported into mitochondria isolated from FL and N+C cells. After indicated time periods, aliquots were removed and Proteinase K (PK) was added where indicated. Samples were analyzed by SDS–PAGE, autoradiography and quantification of PK-protected mature forms of imported proteins. pF1β - precursor of the β subunit of FoF1 ATPase. pcytb2(1–167)△DHFR - precursor consisting of the first 167 residues with the deleted sorting signal of yeast cytochrome b2 fused to mouse dihydrofolate reductase (DHFR); pSu9(1–69)DHFR - matrix targeting signal (residues 1–69) of subunit 9 of FoF1 ATPase from Neurospora crassa fused to DHFR; pOxa1 - precursor of Oxa1; pDLD1 - precursor of D-lactate dehydrogenase; pcytb2 - precursor of cytochrome b2; AAC - precursor of ATP/ADP carrier; p, i, m - precursor, intermediate, and mature forms of imported proteins; * - in vitro translation product starting from an internal methionine. ** - clipped form of Tim23. (H) Membrane potential of isolated mitochondria was measured using DiSC3(5). Valinomycin was added to dissipate membrane potential.