title: Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence.
abstract: Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone being capable of directing non-mitochondrial proteins into mitochondria. These presequences were predicted to form two α helices (α1: N 1-10; α2: N 16-32) separated by loops. Selective deletion of the α1 helix abolished the mitochondrial import of hGDHs. While the α1 helix alone had a very weak hGDH mitochondrial import capacity, it could direct efficiently non-mitochondrial proteins into mitochondria. In contrast, the α2 helix had no autonomous mitochondrial-targeting capacity. A peptide consisting of α1 and α2 helices without intervening sequences had GDH transport efficiency comparable with that of N53. Mutagenesis of the cleavage site blocked the intra-mitochondrial processing of hGDHs, but did not affect their mitochondrial import. Replacement of all three positively charged N-terminal residues (Arg3, Lys7 and Arg13) by Ala abolished import. We conclude that the synergistic interaction of helices α1 and α2 is crucial for the highly efficient import of hGDHs into mitochondria.
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion (IMBB-FORTH), Crete, Greece Department of Biology, University of Crete, Heraklion, Crete, Greece.
authors.forename: Ester
authors.initials: E
authors.lastname: Kalef-Ezra
authors.affiliation: Neurology Laboratory, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Kotzamani
authors.affiliation: Neurology Laboratory, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Zaganas
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion (IMBB-FORTH), Crete, Greece.
authors.forename: Nitsa
authors.initials: N
authors.lastname: Katrakili
authors.affiliation: Neurology Laboratory, School of Health Sciences, Faculty of Medicine, University of Crete, Heraklion, Crete, Greece.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plaitakis
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion (IMBB-FORTH), Crete, Greece Department of Materials Science and Technology, University of Crete, Heraklion, Crete, Greece Institute of Molecular Cell and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K.
authors.forename: Kostas
authors.initials: K
authors.lastname: Tokatlidis
mesh_terms: D003001:Cloning, Molecular; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism
chemical_list: D005969:Glutamate Dehydrogenase
keywords: glutamate dehydrogenase; human; mitochondria; presequence; protein import