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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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 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 |
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Status: | Published |
Refereed: | Yes |
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 > School of Molecular Biosciences |
Journal Name: | Biochemical Journal |
Publisher: | Portland Press Ltd. |
ISSN: | 0264-6021 |
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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