Derepression of citrate synthase in Saccharomyces cerevisiae may occur at the level of transcription.

Abstract

Pulse-chase labeling in whole cells and cell-free protein synthesis were l,ed to The level of mitochondrial enzymes is finely tuned to the energy demands of the cell. In bakers' yeast (Saccharomyces cerevisiae), the synthesis of respiratory chain components and enzymes of the citric acid cycle is repressed in the absence of oxygen or in the presence of glucose (9,18,24,25). The reverse of this process, the derepression of mitochondrial enzymes, is most easily monitored spectroscopically by the reappearance of cytochromes, but the synthesis of many achromatic mitochondrial enzymes is also stimulated by the removal of glucose from the culture medium or by the introduction of air to an anaerobic culture. Recent evidence suggests that synthesis of iso-1-cytochrome c in S. cereivisiae is regulated at the stage of transcription by the availability of heme (10, 30).We have studied the induction of citrate synthase (citrate oxaloacetate lyase, EC 4.1.3.7) during the growth of yeast cells in the presence of glucose. In Nelurospora crassa, this enzyme is made in the cytoplasm as a larger precursor which is processed to a Fmaller mature form as it is imported into the mitochondrion (12). Preliminary reports suggest that a similar mode of synthesis and transport occurs in S. cerevisiae (2). Like many other yeast mitochondrial proteins (21), citrate synthase is made primarily on "free" polyribosomes in the cytoplasm and is transported into the organelle independently of protein synthesis (28). Our interest in this enzyme was stimulated by the report (25) that citrate synthase activity can be derepressed in S. cerevisiae even in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. This result may reflect the presence of a pool of latent enzyme whose activity can be induced by the appropriate stimulus.We have begun by investigating whether the derepression of citrate synthase occurs at the level of transcription. We report that the amount of translatable RNA which encodes a precursor form of citrate synthase increases as the enzyme level is induced.MATERIALS AND METHODS S. cerevisiae strains and growth conditions. Two haploid S. cerevisiae strains were employed in this study. These were D273-1OB (MA To, ATCC 25657) and D273-10B/la, a [rho-] Purification of citrate synthase. Citrate synthase was purified from isolated S. cerevisiae mitochrondria by affinity elution chromatography (5, 19), using a modification of the method of Davies and Scopes (5). We replaced the carboxymethyl cellulose chromatography with a Biorex 70 column in the same buffer system. Nearly all of the loaded citrate synthase activity was bound to the column, and no activity could be measured in either of the wash fractions. Final specific activity of the isolated enzyme was greater than 1,400 U/mg of protein and represented a 150-fold purification over the crude lysate. Enzyme units are defined as micromoles of reduced coenzyme A formed per minute and were assayed by the method of Parvin (22).Antibodies to citrate synthase were ...

Extramitochondrial citrate synthase activity in bakers' yeast.

“…Specifically, the levels of the enzyme are diminished when cells are grown anaerobically or when fermentable carbon sources are present in the medium (21,23). We have demonstrated that the repression of enzyme synthesis is probably attributable to reduced transcription of the citrate synthase gene (14).…”

“…1) was identical to that obtained by Suissa et al (25), who isolated the gene on a different basis. These authors determined the nucleotide sequence of the gene and identified a reading frame which was sufficient to encode a protein having a molecular weight of 5.34 x 104, corresponding to the size of the citrate synthase precursor (1,4,14). Since the protein encoded was homologous to porcine citrate synthase and since the presence of the cloned gene on a high-copynumber plasmid increased mitochondrial citrate synthase activity, it is likely that the clone of Suissa et al and our clone encoded mitochondrial citrate synthase.…”

“…The filters were then treated for immune detection of yeast citrate synthase as described by Towbin et al (26). Antiserum against yeast citrate synthase was prepared in rabbits by using the affinitypurified enzyme described by Hoosein and Lewin (14). Two independent antibody preparations directed against yeast citrate synthase also reacted with the positive clones.…”

“…The monomer is encoded in the nucleus, synthesized in the cytoplasm as a larger precursor, and imported into the mitochondria. Import is associated with the proteolytic removal of an aminoterminal oligopeptide which specifies transit into mitochondria (4,11,14,25). Synthesis of citrate synthase is controlled by the capacity of cells to oxidize nutrients and by the requirements of cells for redox energy.…”

Saccharomyces cerevisiae contains two functional citrate synthase genes.

“…As in other eucaryotic cells, in Saccharomyces cerevisiae all the enzymes of the TCA cycle are encoded in the nucleus and are transported into the mitochondrial matrix (34). Like other proteins imported to the mitochondria, citrate synthase is synthesized as a precursor (54 kilodaltons [kDa]) which is cleaved to a mature form (52 kDa) during import (1,3,19). The structure of the mature citrate synthase dimers from pig heart and chicken heart has been determined by X-ray crystallography, and regions of the enzymes involved in substrate binding and catalysis have been identified (7,29).…”