Functional Characterization and Localization of Acetyl-CoA Hydrolase, Ach1p, in Saccharomyces cerevisiae

Leh-Miauh Buu,Yee-Chun Chen,Fang-Jen S Lee

doi:10.1074/jbc.m213268200

Leh-Miauh Buu, Yee-Chun Chen + Show 1 more

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https://doi.org/10.1074/jbc.m213268200

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Abstract

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.

Highlights

The concentration of acetyl-CoA in cells is primarily regulated by its rate of synthesis and its utilization in various metabolic pathways
The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis
Putative Nucleotide (CoA) Binding Site Is Required for Ach1p Activity—A data base search showed significant homologies among Ach1p and other CoA-transferases, including Schizosaccharomyces pombe ACH1 (SpACH1; 64% identity), N. crassa Acu8 (NcAcu8; 57% identity), E. coli ACH1-like (EcCat1, GenBankTM accession U28377; 38% identity), A. aceti AarC (AarC; 39% identity), Clostridium kluyveri CAT1 (CkCat1, succinylCoA:coenzyme A transferase; 37% identity), and C. kluyveri CAT2 (CkCat2, butyryl-CoA-acetate Coenzyme A; 20% identity)

Summary

Introduction

The concentration of acetyl-CoA in cells is primarily regulated by its rate of synthesis and its utilization in various metabolic pathways. In the yeast Saccharomyces cerevisiae, biosynthesis of acetyl-CoA is mainly achieved by the acetylCoA synthetase reaction, whereas oxidative decarboxylation by the mitochondrial pyruvate dehydrogenase complex appears to be of minor importance (reviewed in Ref. 1). An additional pool of acetylCoA is required for the glyoxylate cycle (citrate synthase and malate synthase reactions) when cells grow with a non-fermentable substrate such as ethanol or acetate. It has been shown that the expression of acetyl-CoA hydrolase (ACH1) from S. cerevisiae is glucose-repressible [15] and subjected to cAMP-dependent repression [16]. An acu-8 mutant strain, characterized as acetate non-utilizing, shows strong growth inhibition by acetate but will use it as a carbon source at low concentrations [20]. We provide initial evidence that Ach1p is involved in development of pseudohyphae but not in mitochondrial biogenesis

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