AVIDIN FUSION PROTEIN AS A TOOL TO GENERATE A STABLE TRANSLOCATION INTERMEDIATE SPANNING THE MITOCHONDRIAL-MEMBRANES

We have designed a fusion gene encoding a chimeric mitochondrial precu rsor protein (avidin fusion protein) that consists of the mitochondria l presequence followed by mouse dihydrofolate reductase, a spacer segm ent, and streptavidin, The avidin fusion protein synthesized in vitro formed a tetramer at the avidin moiety on incubation with biotin durin g or after the translation reaction, The avidin fusion protein purifie d from the Escherichia coil overexpresser cells also formed the tetram er on dilution from 6 M urea into buffer containing biotin, In in vitr o import experiments with isolated yeast mitochondria, the tetramer of the avidin fusion protein became stuck across both mitochondrial memb ranes, with its N-terminal dihydrofolate reductase moiety in the matri x and its C-terminal avidin moiety exposed on the mitochondrial surfac e, Accumulation of the translocation intermediate of the fusion protei n inhibited the import of a mitochondrial precursor protein, and allow ed us to estimate the number of mitochondrial import sites.