Identification and Characterization of MAE1 , the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Abstract

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids

“…However, Eqn (9) yields X lb -II(mt-Prv à mt-malate) 0.70. This rather high value is consistent with the observation that malic enzyme gene expression under anaerobic growth is about threefold increased at the transcriptional and about fourfold increased translational level when compared with the aerobic regime [34]. Obviously, the malic enzyme plays a pivotal role for mt-Prv metabolism in both the aerobic and anaerobic regime.…”

“…In S. cerevisiae, this enzyme can involve either NADH or NADPH as a cofactor [33]. The gene encoding the malic enzyme has recently been identified, and the enzyme was shown to be located exclusively in mitochondria [34]. Pyruvate is transported from the cytosol into the mitochondria by the pyruvate (monocarboxylate) carrier protein [35].…”

Transport of carboxylic acids in yeasts

“…The acid enters the cell, being decarboxylated to pyruvate (Rodriguez & Thornton, 1990;Radler, 1993) by the NAD 1 -dependent malic enzyme. Very distinct K m values for malic enzyme have been estimated, 10 mM for S. pombe, 3.2 mM for Z. bailii and 50 mM for S. cerevisiae (Fuck et al, 1973;Kuczynski & Radler, 1982;Viljoen et al, 1994Viljoen et al, , 1999Boles et al, 1998). Pyruvate is then oxidatively decarboxylated to acetaldehyde, which is reduced to ethanol.…”

Carbohydrate and energy-yielding metabolism in non-conventional yeasts

“…In Z. bailii the enzyme seems constitutive [248], while in S. pombe, transcription of the gene mae2 , encoding malic enzyme is induced when cells are grown in high concentrations of glucose or under anaerobic conditions [249]. It has been suggested that this enzyme may play a role in the provision of pyruvate or in maintaining the redox potential under certain conditions [249,250].…”