Fig. 5. rTOM70 is loosely associated with the 400 kDa complex containing rTOM22 and rTOM40. (A) Interaction of rTOM70 with the import components of the outer membrane as probed by coimmunoprecipitation. The outer membranes were solubilized with 2% digitonin-50 mM NaCl, and aliquots of the supernatant were subjected to immunoprecipitation at 4°C for 3 hours using preimmune, anti-rTOM20, anti-rTOM40 or anti-rTOM70 IgGs. The immunoprecipitates were resolved by SDS-PAGE and the proteins were visualized by immunoblotting with antibodies against the indicated proteins. Inp represents 20% of an aliquot of the supernatant fraction. Other conditions were as described in the Materials and Methods. (B) Analysis of rat TOM proteins with blue native PAGE. Rat liver mitochondrial outer membranes were solubilized with 2% digitonin-containing buffer, and solubilized supernatant was subjected to blue native PAGE as described in the Materials and Methods. Gel slots were excised and subjected to the second dimensional Tricine SDS-PAGE. The gels were analyzed by immunoblotting with the indicated IgGs. Marker proteins used were serum albumin, 66 kDa; lactate dehydrogenase, 140 kDa; catalase, 232 kDa; apoferritin, 440 kDa; and thyroglobulin, 669 kDa. (C) Interaction of rTOM70 with rTOM22 and rTOM20 as revealed by the yeast two-hybrid assay. Host strain Y190 was transformed with two plasmids, one encoding the GAL4 DNA-binding domain (BD) fused to the cytosolic domain of either rTOM20 (BD-rTOM20) or rTOM22 (BD-rTOM22) and the other encoding the GAL4-activating domain (AD) fused to the cytosolic domains of the import components (AD-rTOM20, AD-rTOM22, AD-rTOM70, or AD-OM37). Positive interactions were verified by growth of the transformants on synthetic complete medium without histidine (upper panel) or ß-galactosidase activity (lower panel). As a control, the host strain was transformed with an empty vector encoding AD (pACT2).