Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

ABSTRACT In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but p...

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Autores principales:	Harmen M. van Rossum, Barbara U. Kozak, Matthijs S. Niemeijer, James C. Dykstra, Marijke A. H. Luttik, Jean-Marc G. Daran, Antonius J. A. van Maris, Jack T. Pronk
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a401c1fb546048bca8ec58983af76b6c
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spelling	oai:doaj.org-article:a401c1fb546048bca8ec58983af76b6c2021-11-15T15:50:17ZRequirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol10.1128/mBio.00520-162150-7511https://doaj.org/article/a401c1fb546048bca8ec58983af76b6c2016-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00520-16https://doaj.org/toc/2150-7511ABSTRACT In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. IMPORTANCE This study demonstrates, for the first time, that Saccharomyces cerevisiae can be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeast-based production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose depends on a functional carnitine shuttle, provide valuable models for further functional analysis and engineering of this shuttle in yeast and other eukaryotes.Harmen M. van RossumBarbara U. KozakMatthijs S. NiemeijerJames C. DykstraMarijke A. H. LuttikJean-Marc G. DaranAntonius J. A. van MarisJack T. PronkAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 3 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Harmen M. van Rossum Barbara U. Kozak Matthijs S. Niemeijer James C. Dykstra Marijke A. H. Luttik Jean-Marc G. Daran Antonius J. A. van Maris Jack T. Pronk Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol
description	ABSTRACT In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. IMPORTANCE This study demonstrates, for the first time, that Saccharomyces cerevisiae can be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeast-based production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose depends on a functional carnitine shuttle, provide valuable models for further functional analysis and engineering of this shuttle in yeast and other eukaryotes.
format	article
author	Harmen M. van Rossum Barbara U. Kozak Matthijs S. Niemeijer James C. Dykstra Marijke A. H. Luttik Jean-Marc G. Daran Antonius J. A. van Maris Jack T. Pronk
author_facet	Harmen M. van Rossum Barbara U. Kozak Matthijs S. Niemeijer James C. Dykstra Marijke A. H. Luttik Jean-Marc G. Daran Antonius J. A. van Maris Jack T. Pronk
author_sort	Harmen M. van Rossum
title	Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol
title_short	Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol
title_full	Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol
title_fullStr	Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol
title_full_unstemmed	Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol
title_sort	requirements for carnitine shuttle-mediated translocation of mitochondrial acetyl moieties to the yeast cytosol
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/a401c1fb546048bca8ec58983af76b6c
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Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

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