Membrane potential-driven protein import into mitochondria - The sorting sequence cytochrome b(2) modulates the Delta psi-dependence of translocationof the matrix-targeting sequence

The transport of preproteins into or across the mitochondrial inner membran e requires the membrane potential Delta psi across this membrane. Two roles of Delta psi in the import of cleavable preproteins have been described: a n electrophoretic effect on the positively charged matrix-targeting sequenc es and the activation of the translocase subunit Tim23. We report the unexp ected finding that deletion of a segment within the sorting sequence of cyt ochrome b(2), which is located behind the matrix-targeting sequence, strong ly influenced the Delta psi -dependence of import. The differential Delta p si -dependence was independent of the submitochondrial destination of the p reprotein and was not attributable to the requirement for mitochondrial Hsp 70 or Tim23. With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Delta psi -dependence of import was n ot affected. These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known rol es of Delta psi. Indeed, a charge-neutral amino acid exchange in the hydrop hobic segment of the sorting sequence generated a preprotein with an even b etter import, i.e. one with lower Delta psi -dependence than the wild-type preprotein. The sorting sequence functioned early in the import pathway sin ce it strongly influenced the efficiency of translocation of the matrix-tar geting sequence across the inner membrane. These results suggest a model wh ereby an electrophoretic effect of Delta psi on the matrix-targeting sequen ce is complemented by an import-stimulating activity of the sorting sequenc e.