Membrane Potential-Driven Protein Import into Mitochondria

The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Δy across this membrane. Two roles of Δy in the import of cleavable preproteins have been described: an electrophoretic effect on the positively charged matrix-targeting sequences and the activation of the translocase subunit Tim23. We report the unexpected finding that deletion of a segment within the sorting sequence of cytochrome b ₂ , which is located behind the matrix-targeting sequence, strongly influenced the Δy-dependence of import. The differential Δy-dependence was independent of the submitochondrial destination of the preprotein and was not attributable to the requirement for mitochondrial Hsp70 or Tim23. With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Δy-dependence of import was not affected. These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known roles of Δy. Indeed, a charge-neutral amino acid exchange in the hydrophobic segment of the sorting sequence generated a preprotein with an even better import, i.e. one with lower Δy-dependence than the wild-type preprotein. The sorting sequence functioned early in the import pathway since it strongly influenced the efficiency of translocation of the matrix-targeting sequence across the inner membrane. These results suggest a model whereby an electrophoretic effect of Δy on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.