Carnitine‐dependent metabolic activities in Saccharomyces cerevisiae: three carnitine acetyltransferases are essential in a carnitine‐dependent strain

L‐Carnitine is required for the transfer of activated acyl‐groups across intracellular membranes in eukaryotic organisms. In Saccharomyces cerevisiae, peroxisomal membranes are impermeable to acetyl‐CoA, which is produced in the peroxisome when cells are grown on fatty acids as carbon source. In a reversible reaction catalysed by carnitine acetyltransferases (CATs), activated acetyl groups are transferred to carnitine to form acetylcarnitine which can be shuttled across membranes. Here we describe a mutant selection strategy that specifically selects for mutants affected in carnitine‐dependent metabolic activities. Complementation of three of these mutants resulted in the cloning of three CAT encoding genes: CAT2, coding for the carnitine acetyltransferase associated with the peroxisomes and the mitochondria; YAT1, coding for the carnitine acetyltransferase, which is presumably associated with the outer mitochondrial membrane, and YER024w (YAT2), which encodes a third, previously unidentified carnitine acetyltransferase. The data also show that (a) L‐carnitine and all three CATs are essential for growth on non‐fermentable carbon sources in a strain with a disrupted CIT2 gene; (b) Yat2p contributes significantly to total CAT activity when cells are grown on ethanol; and that (c) the carnitine‐dependent transfer of activated acetyl groups plays a more important role in cellular processes than previously realised. Copyright © 2001 John Wiley & Sons, Ltd.