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

Strains.Table 1 describes the characteristics of the strains used in the present work. The construction of S. cerevisiae strain CLA11-700 (MATα LEU4 LEU9 leu2::LEU2 ura3Δ) has been previously described (20). To obtain the leu4Δ mutant strain (CLA11-701) from CLA11-700, LEU4 was replaced by homologous recombination as previously described (24). A module containing the kanMX4 cassette flanked by 915 bp of the LEU4 5′ untranslated region (UTR) (−915 to −1) and 140 bp of the LEU4 3′ UTR (+1861 to +2001) sequences was amplified with deoxyoligonucleotides G1 and G2 and genomic DNA of strain Y37237 from the EUROSCARF (European Saccharomyces cerevisiae Archive for Functional analysis) collection was used as the template (for the deoxyoligonucleotides used in this study, see Table S1 in the supplemental material). Transformants were selected for G418 resistance (200 mg/liter; Life Technologies) and confirmed by PCR. To obtain a leu9Δ mutant, strain CLA11-700 was transformed with a 2,391-bp PCR product containing the kanMX4 cassette flanked by 720 bp of the LEU9 5′ UTR (−720 to −1) and 117 bp of the LEU9 3′ UTR (+1816 to +1933). The module was amplified from genomic DNA of strain Y32364 from the EUROSCARF collection with deoxyoligonucleotides G3 and G4. Transformants were selected for G418 resistance, generating strain CLA11-702; mutants were PCR verified. A leu4Δ leu9Δ double mutant of strain CLA11-703 was constructed by interrupting LEU4 with URA3 and LEU9 with kanMX4. The URA3 gene was amplified by PCR with deoxyoligonucleotides G5 and G6 on genomic DNA from wild-type strain S288C; once obtained, this PCR module was digested with BamHI, generating cohesive ends, and inserted into a pGEM-T easy vector that contained LEU4 sequence previously linearized with BamHI. The leu4::URA3 construct was then PCR amplified with deoxyoligonucleotides G1 and G2, producing a 3,150-bp DNA fragment that was transformed into strain CLA11-702. Transformants were selected for uracil prototrophy and verified by PCR. The S. cerevisiae leu4Δ leu9Δ double mutant (CLA11-703-2) with nourseothricin and G418 resistance (leu4Δ::natMX4 leu9Δ::kanMX4) was obtained by transforming leu4Δ (CLA11-701) with the clonNAT module obtained by EcoRI digestion of plasmid p4339; this module replaces kanMX4 by homologous recombination (25), generating strain CLA11-701-2 (leu4Δ::natMX4 LEU9 ura3 leu2::LEU2). The LEU9 gene was subsequently replaced by homologous recombination with a module containing the kanMX4 cassette amplified from strain CLA11-702 with deoxyoligonucleotides G7 and G8, generating strain CLA11-703-2.

Construction of strains expressing LEU4 and/or LEU9 from the ENO2 promoter.To fuse the LEU4 and LEU9 coding sequences to the ENO2 promoter, the LEU4 and LEU9 ORFs were amplified with deoxyoligonucleotide pairs G9-G10 and G11-G12, respectively, with genomic DNA from strain CLA11-700 as the template. The resulting modules were flanked by sequences homologous to the 5′ and 3′ UTRs of ENO2 (50 bp upstream from the start codon and 50 bp downstream of the stop codon). The modules were transformed independently into leu4Δ leu9Δ double mutant strain CLA11-703, generating strains CLA11-704 (MATa ENO2pr-LEU4 leu9Δ::kanMX4 leu2::LEU2) and CLA11-705 (MATa leu4Δ::URA3 ENO2pr-LEU9 leu2::LEU2) (Table 1). Transformants were selected for leucine prototrophy and PCR verified. An ENO2pr-LEU4 ENO2pr-LEU9 double mutant was constructed as follows. A PCR module containing ENO2pr-LEU4 was amplified from strain CLA11-704 with deoxyoligonucleotides G13 and G14. This module was used as the template for a second PCR with deoxyoligonucleotides G15 and G16 in order to add flanking regions homologous to the 5′ and 3′ UTRs of the LEU4 gene. The resulting modules contained 50 bp of the upstream sequence of the LEU4 start codon, 742 bp of the 5′ UTR of the ENO2 promoter (−742 to −1), the LEU4 ORF (1,860 bp), 381 bp of the 3′ UTR of ENO2 (+1315 to +1695), and 50 bp downstream of the LEU4 stop codon. This module was used to transform strain CLA11-705. Transformants were selected for 5-fluoroorotic acid resistance, producing strain CLA11-706 (MATa ENO2pr-LEU4 ENO2pr-LEU9 leu2::LEU2 ura3Δ). Correct insertion of the modules was verified by PCR.

Construction of yECitrine-tagged mutant S. cerevisiae strains.Deoxyoligonucleotides were designed on the basis of the ENO1 promoter (G16a, G16b), LEU4, (G17, G18), ENO2 promoter-LEU4 (G17, G21), LEU9 (G19, G20), or ENO2 promoter-LEU9 (G19, G21) carrying 18 bp of yECitrine (forward) or URA3 (reverse) and used to PCR amplify yECitrine and URA3 from plasmid pKT175. The resulting modules were selectively transformed into BY4741, CLA11-704, CLA11-705, and CLA11-706 to generate strains BY4741-700, BY4741-701, BY4741-702, CLA-11-704-1, CLA11-705-1, CLA11-706-1, and CLA11-706-2 (25, 26) and confirmed by PCR.

Plasmid construction for bimolecular fluorescence complementation (BiFC) assay analysis.To analyze interactions between the paralogous proteins Leu4 and Leu9, a pair of plasmids encoding either the N- or C-terminal domain of the yECitrine protein were generated with the QuikChange site-directed mutagenesis kit (Stratagene catalog no. 200518). In order to prepare a plasmid coding for the155 amino acids of the yECitrine N-terminal half (pKT175-yN-CaURA3), we used as the template the vector pKT175 (26) in which a stop codon was generated at position +465 of yECitrine by amplification with deoxyoligonucleotides G22 and G23, both harboring a stop codon. To generate a plasmid that encodes the C-terminal half of the cyan fluorescent protein (pKT101-yC-SpHIS5), we generated a vector with the last 249 bp of the cyan fluorescent protein-encoding gene, which corresponds to the C terminus of this protein (amino acids 156 to 238). This was achieved by amplification of the template pKT101 (26) with deoxyoligonucleotides G24 and G25, which introduce a deletion of 465 bp from the pKT101; this deletion corresponds to the cyan fluorescent protein N terminus (the first 155 amino acids). It is important to mention that when the halves of these two proteins are complemented, yECitrine fluorescence is reconstituted. The resulting plasmids were sequenced with an Applied Biosystems 3100 genetic analyzer.

BiFC analysis.To carry out BiFC analysis tagged Leu4 and Leu9 proteins were prepared as follows. About 2.1-kb-long fragments yN-CaURA3 and yC-SpHIS5 were amplified from plasmids pKT175-yN-CaURA3 (yN) and pKT101-yC-SpHIS5 (yC), respectively, with deoxyoligonucleotides G17 and G18 for LEU4 and G19 and G20 for LEU9. Leu4 and Leu9 were tagged at the C-terminal end with either the yN or the yC half of the fluorescent protein in strains BY4741 MATa and Y8205 MATα (27), which were subsequently mated. As a positive control in the BiFC analysis, the dimeric Bat1 protein was alternatively tagged at the C-terminal end with either the yN or the yC half obtained by PCR amplification with deoxyoligonucleotides G26 and G27 in strains BY4741 MATa and Y8205 MATα. Subsequently, strains were mated and diploid strains were analyzed by confocal microscopy. As a negative control in the BiFC system, strain BY4741 BAT1-yN MATa was mated with a strain Y8205 MATα (27) derivative that contained a mitochondrial yC sequence resulting from the fusion of the first 51 bp, encoding the mitochondrial localization signal from BAT, fused to the yC sequence of yECitrine; this construction was prepared with deoxyoligonucleotides G27 and G28. All diploid strains were analyzed by confocal microscopy.

Construction of plasmids bearing LEU4 or LEU9.Standard molecular biology techniques were used to construct plasmids bearing LEU4 or LEU9 (28). Genes were amplified with their 5′ promoter sequences and cloned into pRS416 (CEN6 ARSH4 URA3). For LEU4, a 3,117-bp region from bp −1087 from the start codon to bp +170 from the stop codon was amplified with deoxyoligonucleotides G57 and G58, generating plasmid pRS416-LEU4. For LEU9, a 3,080-bp region between bp −1120 from the start codon and bp +145 from the stop codon was amplified with deoxyoligonucleotides G59 and G60, generating plasmid pRS416-LEU9. Genes cloned into plasmid pRS416 were subsequently transformed into leu4Δ single mutant and isogenic leu4Δ leu9Δ double mutant S. cerevisiae strains. Yeast strains were transformed by a previously described method (29). Transformants were selected for either uracil prototrophy or leucine prototrophy on minimal medium (MM) and G418 or nourseothricin resistance on yeast extract-peptone-dextrose rich medium and confirmed by PCR.

Growth conditions.S. cerevisiae strains were routinely grown in MM containing salts, trace elements, and vitamins according to the formula for yeast nitrogen base (Difco). Glucose (2%, wt/vol) or ethanol (2%, vol/vol) was used as a carbon source, and 40 mM ammonium sulfate was used as a nitrogen source. Supplements needed to satisfy auxotrophic requirements were added at 0.1 mg/ml. Cells were incubated at 30°C with shaking (250 rpm).

Fluorescence microscopy.To determine the subcellular localization of Leu4 and Leu9-yECitrine, tagged strains BY4741-701 and BY4741-702 were stained with MitoTracker Red CMXRos (Molecular Probes) in accordance with the manufacturer's specifications. Colocalization of MitoTracker and yECitrine was determined by sequential imaging. Confocal images were obtained with a FluoView FV1000 laser confocal system (Olympus) attached to/interfaced with an Olympus IX81 inverted light microscope with a 60× oil immersion objective (UPLASAPO 60× O, numerical aperture 1.35) at a ×20.0 zoom with a 3.5-μm confocal aperture. The excitation and emission wavelengths were as follows: for yECitrine, an excitation wavelength of 488 nm, an emission wavelength of 520 nm, a bright-field wavelength of 500 nm, and a range of 30 nm; for MitoTracker, an excitation wavelength of 543 nm, an emission wavelength of 598 nm, a bright-field wavelength of 555 nm, and a range of 100 nm. Diploid strains BY8205-701, BY8205-702, BY8205-703, and BY8205-704 were stained with MitoTracker Deep Red 633 (Molecular Probes). The subsequent image processing was carried out with Olympus Fluo View FV1000 (version 1.7) software.

Flow cytometry.Strains bearing the LEU4-yECitrine (BY4741-701) and LEU9-yECitrine (BY4741-702) endogenous promoters were grown in MM to exponential growth phase (optical density at 600 nm [OD₆₀₀] of ∼0.6 measured in an LKB Ultrospec III spectrophotometer [Pharmacia] with a 1-cm path length). Strains expressing ENO2pr-LEU4-yECitrine (CLA11-704-1) or ENO2pr-LEU9-yECitrine (CLA11-705-1) from the ENO2 promoter were grown in yeast extract-peptone medium containing 2% galactose (YP-Gal 2%) to an OD₆₀₀ of ∼1.6. Glucose was then added to a final concentration of 10%, and the cells were allowed to grow for 6 h. Collected cells were washed twice with FACSFlow (BD catalog no. 342003) and suspended in this solution to a final OD₆₀₀ of 0.1, and 1 × 10⁴ events were recorded. The measurements were performed with a BD FACSCalibur flow cytometer. The fluorescent signal was obtained with a 488-nm excitation and 530-nm band-pass filter photomultiplier. Data were analyzed offline with the Cyflogic software. For further analysis, we obtained the geometric mean of each set of data as a measurement of protein prevalence as previously reported (30).

α-IPMS enzyme assay and protein determination.For α-IPMS determination, strains expressing LEU4 and/or LEU9 from the ENO2 promoter were grown in YP-Gal 2% to an OD₆₀₀ of ∼1.6, and glucose was added to a final concentration of 10%; samples were collected after 6 h. Strains harboring the endogenous LEU4 and LEU9 promoters were grown to exponential phase (OD₆₀₀ of ∼0.6) in MM with either 2% glucose or ethanol as a carbon source and 40 mM ammonium sulfate as a nitrogen source. Cells were then harvested and suspended in phosphate buffer A (50 mM phosphate buffer [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol). Crude extracts were obtained by disrupting cells with glass beads. α-IPMS activity was assayed with 5 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB; Ellman's reagent) in 1.0 ml of 50 mM phosphate buffer (pH 7.5)–140 mM KCl–4 mM MgCl₂–0.25 mM acetyl-CoA–10 mM α-KIV. After 5 min of thermal equilibration at 30°C and completion of the reaction of DTNB with the contaminant CoA present in the commercial acetyl-CoA preparation, the assay was started by adding cell extract and the initial reaction rate was obtained from the change in absorbance at 412 nm in a Varian Cary 400 spectrophotometer with a 1-cm path length. 2-Nitro-5-thiobenzoate production was quantified by using an extinction coefficient of 14.15 M⁻¹ cm⁻¹ (31). Specific activity is given in nanomoles of CoA formed per minute per milligram of protein. Protein concentration was determined as described previously (32).

Leu4 and Leu9 α-IPMS purification.α-IPMS was purified from extracts prepared from cultures of strains CLA11-704, CLA11-705, and CLA11-706 (Table 1), which overexpress LEU4, LEU9, or both under the control of the ENO2 promoter. Cells were grown in YP-Gal 2% to an OD₆₀₀ of ∼1.6, and glucose was added to a final concentration of 10%; samples were collected after 6 h. Yeast mitochondria were isolated as previously described (33). Mitochondria were harvested by centrifugation and washed twice with cold distilled water, after which samples were disrupted with glass beads in phosphate buffer A. The extract obtained was applied to a Q Sepharose column (Sigma Q1126) previously equilibrated with 50 mM phosphate buffer, pH 7.5. Unbound proteins were eluted with 4 column volumes, and α-IPMS was eluted with 50 mM phosphate buffer, pH 7.5, and a linear gradient of 0.0 to 0.5 M NaCl; fractions showing the highest activity were collected and dialyzed with an Amicon centrifugal filter with a 30-kDa molecular mass cutoff in 50 mM phosphate buffer, pH 7.5.

Molecular mass determination.The native molecular masses of purified α-IPMS isozymes were determined by running the samples on a Superdex 200 HR 10/30 (Amersham Pharmacia Biotech) column equilibrated with 50 mM phosphate buffer (pH 7.5)–0.15 M NaCl. The column was calibrated with the molecular size markers thyroglobulin (670 kDa), alcohol dehydrogenase (150 kDa), and bovine serum albumin (66 kDa) from Sigma. The flow rate in the column was 0.25 ml/min, and 0.25-ml fractions were collected; the eluted fractions were monitored at 280 nm with a Pharmacia fast protein liquid chromatography system.

Native agarose gel.Strains CLA11-704-1, CLA11-705-1, CLA11-706-1, CLA11 706-2, and BY4741-700 were grown in YP-Gal 2% to an OD₆₀₀ of ∼1.6; at that point, glucose was added to a final concentration of 10%. Samples were collected after 6 h, harvested by centrifugation, and washed twice with cold distilled water. Crude protein extracts were obtained by disrupting the cells with glass beads in phosphate buffer A. A horizontal 1% agarose gel (UltraPure LMP Agarose; Gibco catalog no. 5517UA) was prepared in buffer B (25 mM Tris-HCl [pH 8.8], 19.2 mM glycine). The gel was submerged in a reservoir containing buffer B (34), and crude protein extracts (∼ 500 μg) were loaded onto the agarose gel. Electrophoresis was carried out at 4°C at 35 V for 5 h, and yECitrine fluorescence was visualized directly under a UV transilluminator.

Enzyme kinetics and data analysis.Initial velocity measurements were performed by varying both substrates simultaneously (acetyl-CoA at 0.005 to 0.2 mM and α-KIV at 0.01 to 0.25 mM), and the results where globally fitted to equation 1 (see below) with the Prism software. Equation 1 is as follows: v=Vmax [A][B]KiaKb+Kb[A]+Ka[B]+[A][B](1) where v is the initial velocity; V_max is the maximal velocity; A is the acetyl-CoA concentration; B is the α-KIV concentration; Ka and Kb are the K_m values for A and B, respectively; and Kia is the dissociation constant for A. The resulting kinetic parameters are shown in Table 2.

To study leucine inhibition, saturation curves were determined at the following leucine concentrations: 0, 0.04, 0.08, 0.12, and 0.2 mM for Leu4; 0.0, 1.0, 2.0, 3.0, and 4.0 mM for Leu9; and 0.0, 0.25, 0.5, 0.7, and 1.0 mM for the heteromeric isoform. At every leucine concentration, the acetyl-CoA concentration was varied (0.005 to 0.25 mM) while the α-KIV concentration was fixed at 2.5 mM. When the α-KIV concentration was varied (0.0025 to 2.5 mM), the acetyl-CoA concentration was fixed at 0.25 mM. In order to select the inhibition model, the data were fitted to different models, including a cooperative one with the program Dynafit; the best fit obtained corresponded to a mixed inhibition model. Results were globally fitted with the Prism software to equation 2 (see below) when the varied substrate was acetyl-CoA and to equation 3 (see below) when α-KIV was the variable substrate. Equations 2 and 3 are as follows: v=Vmax [A]Ka(1+KibB)(1+IKis)+[A](1+KbB)(1+IKii)(2) v=Vmax [B]Kb(1+KiaA)(1+IKis)+[B](1+KaA)(1+IKii)(3) where I is the leucine concentration; Kis is the slope-associated inhibitor constant, which reflects the extent of leucine dissociation to the free enzyme; and Kii is the intercept-associated inhibitor constant, which indicates the extent of leucine dissociation to the enzyme-substrate complex. The other parameters have the same meanings as in equation 1. The resulting kinetic parameters are also shown in Table 2.

Northern blot analysis.Northern blot analysis was carried out as previously described (35). Total yeast RNA was prepared from 100-ml aliquots of cultures grown to an OD₆₀₀ of ∼0.6 in MM with ammonium sulfate as a nitrogen source and 2% glucose or ethanol as a carbon source. PCR products were used as probes. For LEU4, a 192-bp product was amplified with deoxyoligonucleotides G65 and G66; for LEU9, a 193-bp PCR product was amplified with deoxyoligonucleotides G67 and G68; and a 1,200-bp fragment of ACT1 was used as an internal loading standard. Blots were scanned with the program ImageQuant 5.2 (Molecular Dynamics).

Metabolite extraction and analysis.Cell extracts were prepared from exponentially growing cultures. Samples used for intracellular amino acid determination were treated as previously described (20).