title: Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence.
abstract: The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Deltapsi across this membrane. Two roles of Deltapsi 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 Deltapsi-dependence of import. The differential Deltapsi-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 Deltapsi-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 Deltapsi. 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 Deltapsi-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 Deltapsi on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Geissler
authors.forename: T
authors.initials: T
authors.lastname: Krimmer
authors.forename: U
authors.initials: U
authors.lastname: Bömer
authors.forename: B
authors.initials: B
authors.lastname: Guiard
authors.forename: J
authors.initials: J
authors.lastname: Rassow
authors.forename: N
authors.initials: N
authors.lastname: Pfanner
mesh_terms: D000595:Amino Acid Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008564:Membrane Potentials / Q000502:physiology*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D004792:Enzyme Precursors; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase