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. 2015 Jun;14(6):564-77.
doi: 10.1128/EC.00033-15. Epub 2015 Apr 3.

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae

Geovani López  1 , Héctor Quezada  2 , Mariana Duhne  2 , James González  2 , Mijail Lezama  2 , Mohammed El-Hafidi  3 , Maritrini Colón  2 , Ximena Martínez de la Escalera  2 , Mirelle Citlali Flores-Villegas  2 , Claudio Scazzocchio  4 , Alexander DeLuna  5 , Alicia González  2
Affiliations

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae

Geovani López et al. Eukaryot Cell. 2015 Jun.

Abstract

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

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Figures

FIG 1
FIG 1
Diagrammatic representation of the compartmentalization of the leucine amino acid biosynthetic pathway of S. cerevisiae. The proteins that participate in the pathway are Leu1 (isopropyl malate isomerase), Leu2 (β-IPM dehydrogenase), Leu5 (mitochondrial inner membrane protein, involved in the transport of CoA to the mitochondrial matrix), Ilv2 (acetolactate synthase), Ilv5 (acetohydroxyacid reductoisomerase), Ilv3 (dihydroxyacid dehydratase), Bat1 (mitochondrial branched-chain amino acid aminotransferase), Bat2 (cytosolic branched-chain amino acid aminotransferase), Leu4 (mitochondrial leucine-sensitive α-IPMS), Leu9 (mitochondrial leucine-resistant α-IPMS), Leu4 s (cytosolic leucine-sensitive α-IPMS), Oac1 (mitochondrial inner membrane transporter; transports oxaloacetate, sulfate, thiosulfate, and IPM), CoA, Ac CoA (acetyl-CoA), PYR (pyruvate), AL (acetolactate), DHIV(α,β-dihydroxyisovalerate), KIV (α-KIV), α-IPM (α-IPMS), and β-IPM. The expression of the genes preceded by a plus sign in parentheses is positively regulated by Leu3.
FIG 2
FIG 2
Growth curves of S. cerevisiae LEU4 and LEU9 single and double mutants. Growth curves of LEU4 LEU9, leu4Δ LEU9, LEU4 leu9Δ, and leu4Δ leu9Δ mutant strains in MM with 2% glucose (A) or 2% ethanol (B) as a carbon source with or without (w/o) leucine, as shown. (C) Growth of the LEU4 LEU9/pRS416, leu4Δ leu9Δ/pRS416, leu4Δ leu9Δ/pRS416-LEU4, and leu4Δ leu9Δ pRS416-LEU9 mutant strains cultivated on 2% glucose or ethanol. (D) Growth of the LEU4 LEU9/pRS416, leu4Δ LEU9/pRS416, leu4Δ LEU9/pRS416-LEU4, and leu4Δ LEU9/pRS416-LEU9 mutant strains cultivated on 2% glucose or ethanol. The values presented are the means of at least four experiments ± the standard deviations.
FIG 3
FIG 3
A leu4Δ LEU9 single mutant strain accumulates leucine pools larger than those found in the wild-type strain. Shown are the intracellular concentrations of leucine determined in extracts obtained from yeast cells grown in MM with glucose (2%, wt/vol) or ethanol (2%, vol/vol) and harvested during exponential growth (OD600 of ∼0.6). Cell extracts were prepared as described in Materials and Methods. The values presented are the means of at least three measurements ± the standard deviations.
FIG 4
FIG 4
Leu4 is the predominant isoform in either glucose- or ethanol-grown cultures. (A) α-IPMS activity determined in extracts of cells grown with either glucose or ethanol (2%, wt/vol). (B) Northern blot analysis of total RNA obtained from S. cerevisiae strains was carried out as stated in the text. Cells were grown in MM with either glucose (G) or ethanol (E) as a carbon source. Filters were sequentially probed with the LEU4- and LEU9-specific PCR products. A 1,200-bp fragment of ACT1 was used as an internal loading standard. Three biological replicates were performed, and representative results are shown.
FIG 5
FIG 5
Leu4 and Leu9 are localized in the mitochondria, allowing direct interaction and giving rise to hetero-oligomeric Leu4-Leu9 isozymes. To determine the subcellular localization of Leu4 and Leu9 paralogues, LEU4-yECitrine LEU9 (BY4741-701) and LEU4 LEU9-yECitrine (BY4741-702) mutant strains were grown in MM to exponential phase (OD600 of ∼0.6). Glucose (2%, wt/vol) was used as a carbon source, and 40 mM ammonium sulfate was used as a nitrogen source. The fluorescence of the Leu4-yECitrine or Leu9-yECitrine fusion colocalizes with the MitoTracker signal in rows 1 and 2. Interactions between paralogous proteins were identified through BiFC by confocal microscopy. Fluorescence images of diploid cells expressing C-terminally yN-tagged Leu4 and C-terminally yC-tagged Leu9 interacting are shown in row 3. Row 4 shows the images of the swap constructions. Row 5 shows the interaction of Bat1-yN and Bat1-yC in diploid strain BY8205-703. Row 6 shows the negative control BAT1-yN and BAT1pr-Bat1-17aa-yC. The BY8205-704 diploid strain shows that the halves of yECitrine do not interact by themselves. DIC, differential interference contrast.
FIG 6
FIG 6
Leu4 and Leu9 homodimers and Leu4-Leu9 heterodimers display α-IPMS activity. (A) Northern blot analysis of total RNA obtained from S. cerevisiae strains CLA11-704 ENO2pr-LEU4 leu9Δ and CLA11-705 leu4Δ ENO2pr-LEU9 was carried out. Probes described in Fig. 4B were used. (B) Strains were grown in YP-Gal 2% to an OD600 of ∼1.6, glucose was added to a final concentration of 10% for 6 h, and then α-IPMS activity was determined in the extracts obtained.
FIG 7
FIG 7
Leu4 and Leu9 preferentially organize in heterodimeric Leu4-Leu9 isoforms. (A) Densitometric scan of purified α-IPMS obtained by SDS-PAGE. Lanes: 1, Leu4 from CLA11-704 (ENO2pr-LEU4 leu9Δ); 2, Leu9 from CLA11-705 (leu4Δ ENO2pr-LEU9); 3, monomers from CLA11-706 (ENO2pr-LEU4 ENO2pr-LEU9). Each peak displays an approximate molecular mass of 68 kDa. (B) Electrophoresis of α-IPMS in native agarose gel. Crude extracts from the ENO1pr-yECitrine (lane 1), ENO2pr-LEU4-yECitrine leu9Δ, (lane 2), ENO2pr-LEU4 ENO2pr-LEU9-yECitrine (lane 3), ENO2pr-LEU4-yECitrine ENO2pr-LEU9 (lane 4), and leu4Δ ENO2pr-LEU9-yECitrine (lane 5) mutant strains were loaded and electrophoresed. (C) Analysis of leucine sensitivity (IC50) of crude extracts obtained from the ENO2pr-LEU4 leu9Δ, leu4Δ ENO2pr-LEU9, and ENO2pr-LEU4 ENO2pr-LEU9 mutant strains grown with 10% glucose. Graphs: I, Leu4 homodimer; II, Leu9 homodimer; III, Leu4-Leu9 heterodimer; IV, mixture of equivalent amounts of extracts of the Leu4 and Leu9 homodimers. Results are averages of at least two biological replicates.
FIG 8
FIG 8
The Leu4-Leu9 heterodimer shows a leucine sensitivity intermediate between those of the Leu4-sensitive and Leu9-resistant isoforms. Saturation curves at different leucine concentrations of the Leu4 (A, D) and Leu9 (B, E) homodimers and the Leu4-Leu9 heterodimer (C, F). Continuous lines represent the global fit to equation 2 or 3.
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