(A) Tim17R105A mutant is correctly inserted into the inner membrane. Submitochondrial localization of Tim17, wild type (WT) and R105A mutant (tim17R105A), was analyzed by its accessibility to Proteinase K (PK) in intact mitochondria, upon opening of the outer membrane (+ swelling) and upon solubilization of both mitochondrial membranes (+TX-100). In addition, mitochondria were subjected to carbonate extraction (CE) and fractionated into supernatant (S) and pellet (P) fractions that contain soluble and membrane proteins, respectively. All samples were analyzed by SDS-PAGE followed by immunoblotting with indicated antibodies. Tom70 served as a marker for a membrane-integrated outer membrane protein, Tim50 for a membrane-integrated inner membrane protein exposed to the intermembrane space and Mge1 for a soluble matrix protein. (B) Mitochondria were isolated from wild type (WT) and tim17R105A (R105A) yeast cells grown under permissive conditions in YPD medium at 24°C. In vitro imports of indicated precursor proteins were performed as described in Figure 2—figure supplement 1. (C) Ten-fold serial dilutions of cells were spotted on YPD and YPLac plates and incubated at indicated temperatures. (D) Isolated mitochondria, as indicated, were solubilized in digitonin-containing buffer and cleared lysates were incubated with affinity purified antibodies to Tim17, Tim23 and Tim16 prebound to Protein A-Sepharose. Antibodies from preimmune (PI) serum were used as a negative control. After three washing steps, proteins specifically bound to the beads were eluted with Laemmli buffer. Total (20%) and bound fractions (100%) were analyzed by SDS-PAGE followed by immunoblotting with indicated antibodies. (E) Cells carrying a His-tagged version of Tim17 and a BPA residue at position 106 were UV irradiated, where indicated. Total cell extracts were prepared, diluted with Triton X-100 buffer and incubated with Ni-agarose beads. After three washing steps, specifically bound proteins were eluted. Samples were analyzed by SDS-PAGE and immunoblotting with Tim44 antibodies.