Figure 5. Ubc8 is required for the full assembly of the TOM complex.

(A) Cells were grown in a glycerol medium. After the addition of glucose for 4 h, the mitochondria were isolated and subjected to blue native gel electrophoresis. The indicated proteins were visualized by Western blotting. (B, C) Radiolabeled Tom22 was imported into wild-type and Δubc8 mitochondria for the indicated time periods followed by incubation with proteinase K to remove non-imported precursor proteins. The membrane-inserted, protease-protected fragment of Tom22 (Tom22’) was analyzed by SDS–PAGE and autoradiography. Unspecific cross-reactions of the antibody are indicated with an asterisk (*). For quantification, the signal of imported Tom22 of the longest time period into wild-type mitochondria was set to 100% (control). Quantification shows mean values and standard deviations of seven independent experiments (n = 7). (D) Radiolabeled Tom22 was incubated with mitochondria isolated from wild-type and Δubc8 cells for the times indicated. Assembly of Tom22 into the TOM complex was analyzed by blue native gel electrophoresis and autoradiography. The position of the TOM complex is indicated. Note that the assembly of Tom22 into Δubc8 mitochondria occurs with reduced efficiency. Unspecific cross-reactions of the antibody are indicated with an asterisk (*). (E) Model of Ubc8 function. See text for details.

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