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. 2016 Jun 21;291(33):17345–17359. doi: 10.1074/jbc.M116.738146

FIGURE 7.

FIGURE 7.

J-domain mutation of JC15 results in a phenotype similar to Pam18. A, 10-fold serial dilutions of wild type and mutant cells were spotted onto yeast extract/peptone/dextrose And Leucine drop out media (Leu−) followed by incubation at the indicated temperatures for 48 h. B, expression analysis of TIM23 translocase components in the mutant. Equivalent amounts of wild type and mutant mitochondria were analyzed by SDS-PAGE and immunoblotting using specific antibodies against the translocon members. C, membrane association analysis of JC15 mutant. Mitochondria from wild type and mutant cells were sonicated, and the membrane fraction was separated by ultracentrifugation before electrophoresis. Unfractionated extract (T), soluble supernatant (S) and membrane pellet (P) fractions were immunoblotted using the indicated antibodies to determine the distribution of the respective proteins. D, E, and F, JC15 is critical for mitochondria biogenesis. Yeast cells expressing wild type Pam18, JC15, or JC15 mutant were stained by MitoTracker (D), NAO (E), or JC-1 (F) and subjected to flow cytometry measurements. The amount of dye uptake in case of wild type Pam18 cells was set at 100%. For JC-1 dye, the ratio of JC-1 red to green florescence was calculated and is represented as a bar chart. Valinomycin was used as positive control. Data are represented as n = 3, mean ± S.E. **, p (two tailed) < 0.001; ***, p (two tailed) < 0.0001. G, import of precursor proteins in JC15 mutant. Early log phase cultures of wild type and mutant JC15 yeast cells were subjected to heat shock at 37 °C for 8 h, and cell lysates were analyzed by SDS-PAGE and immunoblotted using specific anti-Hsp60 antibodies; p, precursor form; m, mature form of Hsp60. H and I, Cytb2(47)-DHFR preprotein was imported into wild type or mutant mitochondria at 25 °C for the indicated time points. Reaction mixes were separated on SDS-PAGE and analyzed by immunoblotting and densitometry (I); p, precursor; i, intermediate; m, mature. The amount of m-Cytb2(47)-DHFR in wild type Pam18 mitochondria after the longest import time was set to 100%.