Figure 11.

BART can bind ANT1 independently from ARL2. [³⁵S]-labeled ARL2 and BART were prepared in separate transcription/translation reactions and used, either alone or in combination, in overlay assays of rat (lanes 1–4) or mouse (lanes 5 and 6) brain mitochondria as described under MATERIALS AND METHODS. No signal was detected in the region of the 32-kDa binding protein when using ARL2 alone (lane 1), but signal was clearly evident when using either BART alone (lane 2) or BART in combination with ARL2 in the presence of GTP (lane 3). This activity was effectively competed with unlabeled, recombinant BART (20 μM; lane 4) and was not observed in the ANT1 knockout (KO) mouse (lane 6). The presence of the doublet (most evident in lane 5) has been noted before (Wallace, unpublished observation) and results from effects of Laemmli's sample buffer, probably SDS, on ANT. This experiment was performed at least three times with essentially identical results.