Abstract
Transport of acetyl-CoA between intracellular compartments is mediated by carnitine acetyltransferases (Cats) that reversibly link acetyl units to the carrier molecule carnitine. The genome of the opportunistic pathogenic yeast Candida albicans encodes several (putative) Cats: the peroxisomal and mitochondrial Cat2 isoenzymes encoded by a single gene and the carnitine acetyltransferase homologs Yat1 and Yat2. To determine the contributions of the individual Cats, various carnitine acetyltransferase mutant strains were constructed and subjected to phenotypic and biochemical analyses on different carbon sources. We show that mitochondrial Cat2 is required for the intramitochondrial conversion of acetylcarnitine to acetyl-CoA, which is essential for a functional tricarboxylic acid cycle during growth on oleate, acetate, ethanol, and citrate. Yat1 is cytosolic and contributes to acetyl-CoA transport from the cytosol during growth on ethanol or acetate, but its activity is not required for growth on oleate. Yat2 is also cytosolic, but we were unable to attribute any function to this enzyme. Surprisingly, peroxisomal Cat2 is essential neither for export of acetyl units during growth on oleate nor for the import of acetyl units during growth on acetate or ethanol. Oxidation of fatty acids still takes place in the absence of peroxisomal Cat2, but biomass formation is absent, and the strain displays a growth delay on acetate and ethanol that can be partially rescued by the addition of carnitine. Based on our results, we present a model for the intracellular flow of acetyl units under various growth conditions and the roles of each of the Cats in this process.
Highlights
Compartmentalization is one of the main characteristics of eukaryotic cells, and separation of metabolic pathways to different organelles is thought to convey an advantage over the unicompartmental system of bacteria
Acetylcarnitine Synthesis in the Carnitine Acetyltransferase Mutant Strains—Previously, we have shown that no carnitine acetyltransferase activity could be detected in a C. albicans cat2 null strain lacking both peroxisomal and mitochondrial Cat2 [5], two other proteins with homology to carnitine acetyltransferases are encoded in the genome: Yat1 and Yat2
Based on detailed phenotypic and biochemical analyses of the various mutant strains, we present a model for carnitinedependent transport between the peroxisomal, mitochondrial, and cytosolic compartments in C. albicans (Fig. 6)
Summary
Carnitine acetyltransferase; MTS, mitochondrial targeting signal; YNB, yeast nitrogen base; MCS, multiple cloning site; MES, 4-morpholineethanesulfonic acid; HA, hemagglutinin. Because Mls and the other key enzyme of the glyoxylate cycle (i.e. isocitrate lyase (Icl1)) are peroxisomal in C. albicans [7], growth on C2 carbon sources, such as ethanol and acetate, requires import of acetyl-CoA into peroxisomes. Whether this import is carnitine-dependent is not known. It has been firmly established that the CAT2 gene is indispensable during growth of C. albicans on non-fermentable carbon sources [4, 5], our understanding of the flow of acetyl units between the peroxisomal, cytosolic, and mitochondrial compartments and the individual roles of mitochondrial and peroxisomal Cat, Yat, and Yat in this process is very limited. Based on the biochemical and phenotypic analyses of the constructed mutants, we present a model explaining the flow of acetyl units between the peroxisomal, mitochondrial, and cytosolic compartments in C. albicans and the role of each of the Cats in this process
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