Figure S6. Ubc8 overexpression leads to reduced Tom22 levels and diminished import rates.

(A) Ubc8 mRNA levels of wild-type cells containing either a plasmid for Ubc8 overexpression (WT+Ubc8) or the empty vector (WT+ev) for control were analyzed by qPCR. Shown is the UBC8 expression in the overexpression strain relative to the expression in the empty vector control. Mean values and standard deviations of three biological replicates (n = 3) are shown. Significance testing was performed with a t test (***P ≤ 0.001). (B) Wild-type cells overexpressing Ubc8 or containing an empty vector were grown in galactose medium to the mid-log phase. 10-fold serial dilutions were dropped onto plates containing glucose, galactose, or glycerol as carbon source. (C, D) Cells were grown in galactose medium, and mitochondria were isolated and subjected to SDS–PAGE and Western blotting using antibodies against Tom22 and Por1 for control. Panel (D) shows mean values and standard deviations of three biological replicates (n = 3). (E) Radiolabeled Oxa1 was incubated with isolated mitochondria of the indicated strains for different times. Non-imported proteins were removed by the addition of proteinase K. 20% of the radiolabeled proteins used per import reaction were loaded for control. Proteins were visualized by autoradiography. For the experiments shown in this figure, cultures were grown in galactose-based medium unless indicated otherwise.

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