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 of YKL029c/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 beta-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.

FIG. 1

Possible role of pyruvate kinase and malic enzyme in the formation of pyruvate as a biosynthetic precursor in S. cerevisiae. Abbreviations: PYK, pyruvate kinase; PCK, phosphoenolpyruvate carboxykinase; MAE, malic enzyme; acetyl-CoA, acetyl coenzyme A; TCA, tricarboxylic acid.

FIG. 2

Sequence identity dendrogram of malic enzymes and related proteins from different organisms. The dendrogram was obtained by comparison of the deduced amino acid sequence of S. cerevisiae Mae1p against all nucleotide sequence databases (December 1997) dynamically translated in all reading frames; partial malic enzyme sequences were omitted. The sequences were clustered by the PC/GENE program (IntelliGenetics Inc., release 6.70, 1992) at standard settings. Blocks of homology are boxed and numbered I to VII. For simplicity, not all sequences of cluster VII are shown. If different malic enzyme-related sequences have been identified in an organism, this is indicated by the numbers or names in parentheses. In this case, if known, also the corresponding intracellular localization is indicated: c, cytoplasm; m, mitochondria; ch, chloroplasts.

FIG. 3

Northern analysis of MAE1 transcription. Cells of the wild-type strain EBY.D149 (lanes 1 to 7) and the mae1 deletion strain EBY.D152 (lane 8) were grown in carbon-limited chemostat cultures (continuous cultures) or in shake flask cultures (batch cultures) with different carbon sources (glucose or ethanol [EtOH]), with different nitrogen sources (ammonia or, in lane 2, aspartate), or supplemented with alanine (lane 7), either aerobically or anaerobically (lane 4). The mae1Δ strain was grown on YPD medium. Total RNA was prepared from these cultures. The filter was probed with a mixture of a 2.1-kb PCR fragment of the MAE1 coding region and a 3.2-kb DNA fragment containing the complete actin sequence (loading control), both labeled with ³²P, in a 1:1 count ratio. The autoradiogram was exposed to Fuji RX.