title: Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria.
abstract: Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase. Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.
authors.affiliation: Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, The University of Western Australia, Crawley 6009, WA, Australia.
authors.forename: Ryan
authors.initials: R
authors.lastname: Lister
authors.forename: Brett
authors.initials: B
authors.lastname: Mowday
authors.forename: James
authors.initials: J
authors.lastname: Whelan
authors.forename: A Harvey
authors.initials: AH
authors.lastname: Millar
mesh_terms: D001692:Biological Transport; D002104:Cadmium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011198:Solanum tuberosum / Q000166:cytology; D013997:Time Factors; D017967:Zinc Compounds / Q000494:pharmacology*
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D033681:Mitochondrial Membrane Transport Proteins; D010618:Phenanthrolines; D010940:Plant Proteins; D017967:Zinc Compounds; D002104:Cadmium; C016837:zinc chloride; D004492:Edetic Acid; C025205:1,10-phenanthroline