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

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 isoenzyme...

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Autores principales: Kalef-Ezra, Ester, Kotzamani, Dimitra, Zaganas, Ioannis, Katrakili, Nitsa, Plaitakis, Andreas, Tokatlidis, Kostas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095901/
https://www.ncbi.nlm.nih.gov/pubmed/27422783
http://dx.doi.org/10.1042/BCJ20160535
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author Kalef-Ezra, Ester
Kotzamani, Dimitra
Zaganas, Ioannis
Katrakili, Nitsa
Plaitakis, Andreas
Tokatlidis, Kostas
author_facet Kalef-Ezra, Ester
Kotzamani, Dimitra
Zaganas, Ioannis
Katrakili, Nitsa
Plaitakis, Andreas
Tokatlidis, Kostas
author_sort Kalef-Ezra, Ester
collection PubMed
description 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.
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spelling pubmed-50959012016-11-08 Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence Kalef-Ezra, Ester Kotzamani, Dimitra Zaganas, Ioannis Katrakili, Nitsa Plaitakis, Andreas Tokatlidis, Kostas Biochem J Research Articles 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. Portland Press Ltd. 2016-09-15 2016-09-12 /pmc/articles/PMC5095901/ /pubmed/27422783 http://dx.doi.org/10.1042/BCJ20160535 Text en © 2016 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0) .
spellingShingle Research Articles
Kalef-Ezra, Ester
Kotzamani, Dimitra
Zaganas, Ioannis
Katrakili, Nitsa
Plaitakis, Andreas
Tokatlidis, Kostas
Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
title Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
title_full Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
title_fullStr Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
title_full_unstemmed Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
title_short Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
title_sort import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095901/
https://www.ncbi.nlm.nih.gov/pubmed/27422783
http://dx.doi.org/10.1042/BCJ20160535
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