Figure 6.

Import and assembly of non-Tom outer membrane precursors occurs independently of the SAM complex. (A) ³⁵S-labeled precursors of Fis1 (top) and Gem1 (bottom) were imported into mitochondria from wild-type (WT), sam50-1, sam35-2, and sam37Δ yeast cells for the indicated times. After, import-isolated mitochondria were treated with 0.1 M Na₂CO₃, pH 11.5, for 30 min on ice. Membranes were isolated by ultracentrifugation, solubilized in laemmli buffer, and analyzed by Tris-tricine gel electrophoresis and digital autoradiography. (B) ³⁵S-labeled OM45 precursor was imported as described in A. Mitochondria were solubilized in digitonin-containing buffer for blue native electrophoresis (top) or treated with 0.1 M Na₂CO₃, pH 11.5, for SDS-PAGE analysis (bottom). (C) ³⁵S-labeled Fzo1 precursor was imported, and samples were treated as described for A. (D) The ³⁵S-labeled precursor of Ugo1 was imported as described for A. Mitochondria were solubilized in digitonin-containing buffer for blue native electrophoresis (top) or treated with 0.1 M Na₂CO₃, pH 11.5, for SDS-PAGE analysis (bottom).