C- to N-terminal translocation of preproteins into mitochondria

Nuclear-encoded mitochondrial matrix proteins in most cases contain N-termi nal targeting signals and are imported in a linear N- to C-terminaI (N-->C) fashion. We asked whether import can also occur in a C- to N-terminal dire ction (C-->N). We placed targeting signals at the C-terminus of passenger p roteins. Import did occur in this 'backwards' fashion. It paralleled that o f the 'normal' N-->C mechanism in terms of efficiency, rate, energetic requ irements and ability to mediate unfolding and refolding during and followin g import of protein containing a folded domain. Furthermore, this reaction was mediated by the TIM17-23 machinery. The import pathway taken by certain inner-membrane proteins contains elements of such a C-->N translocation pa thway, as they are targeted to mitochondria by internal targeting signals, These internal targeting signals appear to form loop structures together wi th neighbouring transmembrane segments, and penetrate the inner membrane in a membrane-potential-dependent manner. The dimeric TIM17-23 complex, toget her with mt-Hsp70, acts on both sides of the loop structure to facilitate t heir translocation into the matrix. On one side of the loop import occurs i n the common N-->C direction, whereas the translocation of the other side i nvolves the novel C-->N import direction. We conclude therefore that the mi tochondrial import machinery displays no preference for the directionality of the import process.