Translation-coupled translocation of yeast fumarase into mitochondria in vivo.

Fumarase represents proteins that cannot be imported into mitochondria after the termination of translation (post-translationally). Utilizing mitochondrial and cytosolic versions of the tobacco etch virus (TEV) protease, we show that mitochondrially targeted fumarase harboring a TEV protease recognition sequence is efficiently cleaved by the mitochondrial but not by the cytosolic TEV protease. Nonetheless, fumarase was readily cleaved by cytosolic TEV when its import into mitochondria was slowed down by either (i) disrupting the activity of the TOM complex, (ii) lowering the growth temperature, or (iii) reducing the inner membrane electrochemical potential. Accessibility of the fumarase nascent chain to TEV protease under such conditions was prevented by low cycloheximide concentrations, which impede translation. In addition, depletion of the ribosome-associated nascent polypeptide-associated complex (NAC) reduced the fumarase rate of translocation into mitochondria and exposed it to TEV cleavage in the cytosol. These results indicate that cytosolic exposure of the fumarase nascent chain depends on both translocation and translation rates, allowing us to discuss the possibility that import of fumarase into mitochondria occurs while the ribosome is still attached to the nascent chain.