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Methods. 2017 Jan 15;113:111-119. doi: 10.1016/j.ymeth.2016.10.012. Epub 2016 Oct 26.

Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.

Author information

1
Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC, UMR 7178, F-67000 Strasbourg, France.
2
Plateforme Protéomique Strasbourg - Esplanade, Institut de Biologie Moléculaire et Cellulaire, CNRS, FRC 1589, 15 rue Descartes, F-67084 Strasbourg Cedex, France.
3
Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France.
4
Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UGA 5249, iRTSV/LCBM, CEA Grenoble, Grenoble, France.
5
Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France. Electronic address: M.Sissler@ibmc-cnrs.unistra.fr.

Abstract

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.

KEYWORDS:

Aminoacyl-tRNA synthetases; Human; Maturation; Mitochondria; Mitochondrial targeting sequence; N-terminomics; Shotgun proteomics

PMID:
27793688
DOI:
10.1016/j.ymeth.2016.10.012
[Indexed for MEDLINE]
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