Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence

Kalef-Ezra, E., Kotzamani, D., Zaganas, I., Katrakili, N., Plaitakis, A. and Tokatlidis, K. (2016) Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence. Biochemical Journal, 473(18), pp. 2813-2829. (doi: 10.1042/BCJ20160535) (PMID:27422783) (PMCID:PMC5095901)

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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 alpha 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 to 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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Tokatlidis, Professor Kostas
Authors: Kalef-Ezra, E., Kotzamani, D., Zaganas, I., Katrakili, N., Plaitakis, A., and Tokatlidis, K.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Biochemical Journal
Publisher:Portland Press Ltd.
ISSN (Online):1470-8728
Published Online:15 July 2016
Copyright Holders:Copyright © 2016 The Authors
First Published:First published in Biochemical Journal 2016
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
635181SULSA Chair in Systems Biology - Cathcart ChairRichard CogdellScottish Funding Council (SFC)HR07019RI MOLECULAR CELL & SYSTEMS BIOLOGY
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617601Next generation sequencing (NGS) approaches to genotyping in myotonic dystrophy type 1 (ISSF Catalyst Fund)Darren MoncktonWellcome Trust (WELLCOME)097821/Z/11/ZRI MOLECULAR CELL & SYSTEMS BIOLOGY