Requirements for carnitine-shuttle-mediated translocation of mitochondrial acetyl moieties to the Saccharomyces cerevisiae cytosol. Saccharomyces cerevisiae strain:IMX745

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 (IMX745). Laboratory evolution yielded mutants of this strain 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 (IMS0482, IMS0483). Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty-acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2) and encoding an L-carnitine acetyl-transferase (YAT2). Introduction of these mutations into the non-evolved parental strain enabled L-carnitine-dependent growth on glucose. This study identifies 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.