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Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein

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Abstract

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD+. After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.

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Abbreviations

SIRT:

Sirtuin

BSA:

Bovine serum albumin

PTM:

Post-translational modification

CACT:

Carnitine/acylcarnitine transporter

LCAD:

Long-chain acyl-CoA dehydrogenase

CPT1:

Carnitine palmitoyltransferase I

CAT:

Carnitine O-acetyltransferase

GSH:

l-Glutathione reduced

GSSG:

l-Glutathione oxidized

AAC:

ADP/ATP carrier

NAM:

Nicotinamide

PVDF:

Polyvinyldifluoride membrane

NAD+ :

Nicotinamide adenine dinucleotide

NEM:

N-Ethylmaleimide

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Acknowledgements

This work was supported by funds from: Programma Operativo Nazionale [01_00937] - MIUR “Modelli sperimentali biotecnologici integrati per lo sviluppo e la selezione di molecole di interesse per la salute dell’uomo”.

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    Authors and Affiliations

    1. CNR Institute of Biomembranes and Bioenergetics, Via Amendola 165/A, 70126, Bari, Italy

      Nicola Giangregorio & Annamaria Tonazzi

    2. Department DiBEST (Biologia, Ecologia, Scienze Della Terra), Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via Bucci cubo 4C, 87036, Arcavacata di Rende, CS, Italy

      Lara Console & Cesare Indiveri

    3. Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy

      Nicola Giangregorio & Annamaria Tonazzi

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    1. Nicola Giangregorio
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    Correspondence to Nicola Giangregorio or Cesare Indiveri.

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    The experiments comply with the current Italian laws.

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    Nicola Giangregorio and Annamaria Tonazzi have contributed equally to this work.

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    Giangregorio, N., Tonazzi, A., Console, L. et al. Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein. Mol Cell Biochem 426, 65–73 (2017). https://doi.org/10.1007/s11010-016-2881-0

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