In Kluyveromyces lactis a Pair of Paralogous Isozymes Catalyze the First Committed Step of Leucine Biosynthesis in Either the Mitochondria or the Cytosol

Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of Saccharomyces cerevisiae (S. cerevisiae) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as Kluyveromyces lactis (K. lactis) remain to be addressed. The genome of K. lactis, an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the KlLEU4 and KlLEU4BIS paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the S. cerevisiae ScLEU4/ScLEU9 paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in ScLeu4-ScLeu4 and ScLeu4-ScLeu9 sensitive dimers and ScLeu9-ScLeu9 relatively resistant homodimers. Previous work has shown that, in a Scleu4Δ mutant, ScLEU9 expression is increased and assembly of ScLeu9-ScLeu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) K. lactis harbors a sporadic gene duplication, comprising the KlLEU4, syntenic with S. cerevisiae ScLEU4 and ScLEU9, and the non-syntenic KlLEU4BIS, arising from a pre-WGD event. (ii) That both, KlLEU4 and KlLEU4BIS encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (KlLeu4) and the cytosol (KlLeu4BIS), respectively. (iii) That both, KlLEU4 or KlLEU4BIS complement the Scleu4Δ Scleu9Δ leucine auxotrophic phenotype and revert the enhanced ScLEU9 transcription observed in a Scleu4Δ ScLEU9 mutant. The Scleu4Δ ScLEU9 growth mutant phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4Δ Scleu9Δ leucine auxotrophy and the Scleu4Δ ScLEU9 complex phenotype.

Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of Saccharomyces cerevisiae (S. cerevisiae) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as Kluyveromyces lactis (K. lactis) remain to be addressed. The genome of K. lactis, an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the KlLEU4 and KlLEU4BIS paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the S. cerevisiae ScLEU4/ScLEU9 paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in ScLeu4-ScLeu4 and ScLeu4-ScLeu9 sensitive dimers and ScLeu9-ScLeu9 relatively resistant homodimers. Previous work has shown that, in a Scleu4 mutant, ScLEU9 expression is increased and assembly of ScLeu9-ScLeu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) K. lactis harbors a sporadic gene duplication, comprising the KlLEU4, syntenic with S. cerevisiae ScLEU4 and ScLEU9, and the non-syntenic KlLEU4BIS, arising from a pre-WGD event. (ii) That both, KlLEU4 and KlLEU4BIS encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (KlLeu4) and the cytosol (KlLeu4BIS), respectively. (iii) That both, KlLEU4 or KlLEU4BIS complement the Scleu4 Scleu9 leucine auxotrophic phenotype and revert the enhanced ScLEU9 transcription observed in a Scleu4 ScLEU9 mutant. The Scleu4 ScLEU9 growth mutant INTRODUCTION Gene duplication is a source of new or specialized biological functions (Ohno, 1970;Lynch et al., 1991;Force et al., 1999;Kellis et al., 2004). Retained duplicate genes (paralogs) can provide increased dosage of the same product, or may go through a process of sub-or neo-functionalization. In the former, both copies of the gene lose and/or specialize a subset of their ancestral functions, in the latter, at least one of the copies acquires a new function (Ohno, 1970;Lynch et al., 1991;Force et al., 1999).
Paralogous genes can originate from Whole Genome Duplication (WGD) (ohnologs) events or from sporadic duplications known as Small Scale Duplications (SSD) (paralogs). The extant yeast genomes from the Saccharomyces and closely related genera arose from a WGD event within the Saccharomycetaceae family (Kellis et al., 2004). More recent work established that this duplication originated from the fusion of two different species genomes (Marcet-Houben and Gabaldón, 2015). The lineage that gave rise to Kluyveromyces lactis (K. lactis) diverged from the Saccharomyces cerevisiae (S. cerevisiae) lineage before the WGD event (Kellis et al., 2004). Accordingly, this yeast exhibits much less overall genetic redundancy (Conde e Silva et al., 2009), suggesting that the paralogs genes present in K. lactis originated from independent SSD events (Dujon et al., 2004;Huerta-Cepas et al., 2014). Subfunctionalization of paralogs pairs can be achieved through various non-exclusive molecular mechanisms such as modifications of the coding sequence leading to: (i) changes in the kinetic parameters (DeLuna et al., 2001;Rojas-Ortega et al., 2018), (ii) differential subcellular localization (Marques et al., 2008), (iii) formation of hetero-oligomeric isozymes with emerging biochemical properties (DeLuna et al., 2001;López et al., 2015), or (iv) modifications of the regulatory region determining differential expression of each copy (Avendaño et al., 2005;Quezada et al., 2008;González et al., 2017).
The analysis of the functional diversification of orthologous gene pairs from S. cerevisiae and K. lactis could allow addressing the question of whether functional diversification of a given paralogous pair follows either the same or distinct diversification pathways in different organisms. An interesting example of duplicated pairs, present in both S. cerevisiae and K. lactis is that of α-isopropylmalate synthases (α-IPMSs). The ScLEU4 (YNL104C) and ScLEU9 (YOR108W) genes from S. cerevisiae form part of a duplicated chromosomal block generated from the WGD event (Wolfe and Shields, 1997), while those present in the K. lactis KlLEU4 (KLLA0F23529G alias KLLA-ORF403) and KlLEU4BIS (KLLLA0D14201G alias KLLA-ORF4593) were originated from an independent gene duplication event prior to the WGD (see below).
In S. cerevisiae, the leucine biosynthesis intermediate α-IPM plays a dual physiological role. On the one hand, it is the leucine precursor (Chang et al., 1985;Kohlhaw, 2003), on the other, it acts as the co-activator of the ScLeu3 master transcriptional regulator (Kohlhaw, 2003), which modulates the expression of a number of genes within and beyond leucine metabolism (Kohlhaw, 2003). ScLeu3 has a dual function (Kohlhaw, 2003). At high α-IPM concentrations it acts as a transcriptional activator of genes involved in branched chain amino acids (Kohlhaw, 2003) and glutamic acid (Hu et al., 1995) biosynthesis. Low intracellular concentrations of α-IPM shift its function to that of a transcriptional repressor of other genes (Kohlhaw, 2003). ORF KLLA0D10593 probably encodes a ScLeu3 K. lactis orthologous protein (KlLeu3).
of heterodimers occurs since both ScLeu4 and ScLeu9 are located in the mitochondria. The ScLeu4-ScLeu4 isoform is inhibited by low leucine concentrations with an inhibition constant (Kii) of 0.22 mM, while the ScLeu9-ScLeu9 homodimer could be considered as leucine resistant, with a 5.44 mM Kii, thus it is only inhibited by high intracellular concentration of this amino acid. The ScLeu4-ScLeu9 hetero-oligomeric isozyme displays a leucine inhibition constant (Kii 0.70 mM) intermediate to that found for the homo-dimeric isozymes (López et al., 2015).
In a Scleu4 ScLEU9 strain, the exclusive presence of the ScLeu9-ScLeu9 leucine-resistant homodimer, results in a growth impaired phenotype, most probably due to the metabolic imbalance produced by the draining of acetyl-CoA to α-IPM and leucine biosynthesis with the consequent depletion of other tricarboxilic acid cycle intermediates (López et al., 2015). This particular phenotype is enhanced by ScLEU9 overexpression in a Scleu4 background (López et al., 2015). These results indicate that in S. cerevisiae, retention and further diversification of the two α-IPMSs has resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway through the different feedback sensitivity of homomeric and heterodimeric isoforms. Here we address whether retention and further sub-functionalization of KlLEU4 and KlLEU4BIS paralogs has led to diversification of their physiological roles. We analyze the kinetic properties and sub-cellular localization of the two paralogs and their ability to complement the cognate mutations in S. cerevisiae.

Strain Transformation
S. cerevisiae and K. lactis strains used in this work are described in Table 1. Yeast strains were transformed following a previously described method (Ito et al., 1983). Transformants were selected for either uracil prototrophy or leucine prototrophy on minimal medium (MM), G418 and/or nourseothricin resistance on yeast extract-peptone-dextrose (YPD)-rich medium and confirmed by PCR.

Growth Conditions
S. cerevisiae and K. lactis strains were pre-grown overnight (16 h) in rich media YPD. Yeast strains were routinely grown on MM containing salts, trace elements, and vitamins following the formula of yeast nitrogen base (Difco). Glucose (2%, w/v) or ethanol (2%, v/v) were used as carbon sources, and 40 mM ammonium sulfate was used as nitrogen source. Supplements needed to satisfy auxotrophic requirements were added at 0.1 mg/mL. Cells were incubated at 30 o C with shaking (250 rpm). Table 1 describes the genotypes of the strains used in the present work. The single Klleu4 mutant strain (K. lactis 155-2) was constructed as follows: KlLEU4 (KLLA0F23529g) was replaced by homologous recombination in strain K. lactis 155-1 using a module containing the kanMX4 cassette flanked by 676 bp of 5 untranslated region (UTR) (−676 to −1) and 474 bp of 3 UTR (+ 1831 to + 2305) sequences of KlLEU4. This module was amplified by overlapped extension PCR with deoxyoligonucleotides A3 and A4 (Supplementary Table S1) using a template built up by three independent modules: (i) the KlLEU4 5 UTR amplified using A3 and A5 deoxyoligonucleotides, (ii) the kanMX4 module flanked by homologous regions of the 5 UTR and 3 UTR of the KlLEU4 gene, which was amplified from pFA6a plasmid using deoxyoligonucleotides A6 and A7, and (iii) the KlLEU4 3 UTR amplified using deoxyoligonucleotides A8 and A4 (Supplementary Table S1). The PCR product was transformed into the K. lactis 155-1 strain. Transformants were selected for G418 resistance. The single Klleu4bis mutant strain (K. lactis 155-3) was generated as described for Klleu4 . Three PCR products were amplified as follows: the one corresponding to (i) the 5 UTR of KlLEU4BIS (KLLA0D14201g) was amplified using deoxyoligonucleotides A9 and A10, (ii) the kanMX4 module flanked by homologous regions to the 5 and 3 UTR of KlLEU4BIS was amplified from pFA6a plasmid using deoxyoligonucleotides A11 and A12, and (iii) the 3 UTR of KlLEU4BIS amplified using deoxyoligonucleotides A13 and A14, were used as template for an overlapped extension PCR using deoxyoligonucleotides A9 and A14. The assembled PCR product contained the

KlLEU4BIS-yECitrine
To construct S. cerevisiae mutant strains harboring either KlLEU4-yECitrine or KlLEU4BIS-yECitrine (Table 1), we used Scleu4 :kanMX4 ScLEU9 (CLA11-701) mutant to replace the kanMX4 cassette for either the KlLEU4-yECitrine or KlLEU4BIS-yECitrine generating Scleu4 :KlLEU4-yECitrine ScLEU9 (CLA11-701-3) and Scleu4 :KlLEU4BIS-yECitrine ScLEU9 (CLA11-701-4) mutant strains. To obtain Scleu4 :KlLEU4-yECitrine LEU9, the KlLEU4-yECitrine tagged gene (4409 bp) was PCR amplified from the genomic DNA obtained from the CBS2359 ku80 -1 strain using the A23 and A24 deoxyoligonucleotide pair (Supplementary Table S1). Using this amplicon as a template, a subsequent PCR was performed using A25 and A26 deoxyoligonucleotides in order to obtain a module (4529 bp) whose 5 end contains an homologous region with ScLEU4 promoter from −150 to −91 and in the 3 end there is an homologous region with 3 UTR of ScLEU4 from +1951 to +2010. This module was used to replace kanMX4 cassette from the strain CLA11-701 (Table 1). Transformants were selected for uracil prototrophy and confirmed by PCR using A27 and A28 deoxyoligonucleotides, the non-recombinants generated a 2330 bp fragment, while in the transformants a 5048 bp fragment was amplified. Same strategy was followed to obtain Scleu4 :KlLEU4BIS-yECitrine ScLEU9. To amplify KlLEU4BIS-yECitrine tagged gene (4468 bp) deoxyoligonucleotides A29 and A30 were used with a genomic DNA obtained from CBS2359 ku80 -2 strain. Subsequently, a second PCR was carried out using this fragment as a template, for this case primers A31 and A32 were used in order to generate an amplicon (4588 bp) whose 5 end is homologous to ScLEU4 promoter from −150 to −91 and the 3 end is homolog to the ScLEU4 3 UTR from +1951 to +2010. This module was used to replace kanMX4 cassette from strain CLA11-701. In this case confirmation was carried out by fluorescent microscopy.

Construction of YEpKD352 Plasmids
Harboring KlLEU4, KlLEU4BIS, ScLEU4, and ScLEU9 Standard molecular biology techniques were followed as previously described (Sambrook et al., 1989). Genes were amplified with their 5 UTR sequence and cloned into plasmid YEpKD352 (pKD1 or URA3). For KlLEU4, a 2984 bp region between −933 from the start codon and + 221 from the stop codon was amplified with deoxyoligonucleotides A33 and A34 (Supplementary Table S1). For KlLEU4BIS, a 2917 bp region at −1000 bp from the start codon and + 165 from the stop codon was amplified with deoxyoligonucleotides A35 and A36. PCR modules were digested and cloned in order to generate the plasmids YEpKD352-KlLEU4 and YEpKD352-KlLEU4BIS. Genes cloned in YEpKD352 were transformed into strain K. lactis 155-4 or in S. cerevisiae strains CLA1-2-4 and CLA1-2-4-9. Transformants were selected for either uracil prototrophy or leucine prototrophy on minimal medium (MM).

Fluorescent Microscopy
To confirm mitochondrial localization of KlLeu4, KlLeu4-yECitrine tagged strain was stained with MitoTracker Red CMXRos (Molecular Probes) according to manufacturer's specifications. Co-localization between the MitoTracker and yECitrine was determined through sequential imaging. Confocal images were obtained using a FluoView FV1000 laser confocal system (Olympus) attached/interfaced to an Olympus IX81 inverted light microscope with a 60x oil-immersion objective (UPLASAPO 60x O NA:1.35), zoom x20.0 and 3.5 µm of confocal aperture. The excitation and emission settings were as follows: yECitrine excitation at 488 nm; emission 520 nm BF 500 nm range 30 nm; MitoTracker excitation 543 nm; emission 598 nm, BF 555 nm range 100 nm. The subsequent image processing was carried out with Olympus Fluo View FV1000 (version 1.7) software.

Northern Blot Analysis
Northern blot analysis was carried out as previously described (Struhl and Davis, 1981). Total yeast RNA was prepared from 100 mL aliquots of cultures grown to an optical density at 600 nm (OD 600nm ) ∼0.6 on MM with ammonium sulfate as nitrogen source and glucose (2%, w/v) or ethanol (2%, v/v) as carbon source. PCR products were used as probes, for KlLEU4 a 1793 bp product was amplified with deoxyoligonucleotides A43 and A44, for KlLEU4BIS a 1644 bp product was amplified with deoxyoligonucleotides A45 and A46 (Supplementary Table S1). As internal loading standard, a probe of 477 bp from 18S was PCR amplified with deoxyoligonucleotides A37 and A38. For ScLEU4, an 1860 bp product was amplified with deoxyoligonucleotides A39 and A40. For ScLEU9, an 1815 bp PCR product was amplified with deoxyoligonucleotides A41 and A42. A 877 bp ScACT1 fragment amplified with deoxyoligonucleotides A47 and A48 was used as internal loading standard. Blots were scanned using the program ImageQuant 5.2 (Molecular Dynamics).
K. lactisα-IPMSs Purification: KlLeu4 and KlLeu4BIS Overexpression in E. coli For KlLeu4 (KLLA0F23529) heterologous expression, the Bl21 (DE3) E. coli strain (Novagen) was transformed. KlLeu4 selected clones were grown in LB medium with 50 mg mL −1 of kanamycin, grown at 30 o C with shaking (250 rpm). When cultures reached an OD 600nm of 0.4, expression of KlLEU4 was induced with 400 mmol L −1 of isopropyl-β-dthiogalactopyranoside (IPTG), incubated overnight at 16 o C with shaking (250 rpm), harvested by centrifugation at 1100 g for 15 min, and the cellular pellet was stored at −70 o C until used.
KlLeu4BIS selected clones were grown in LB medium with 50 mg mL −1 of kanamycin and 70 mg mL −1 of chloramphenicol, grown at 30 o C with shaking (250 rpm). When cultures reached an OD 600nm of 0.6, expression of KlLeu4BIS was induced with 400 mmol L −1 of IPTG, incubated overnight at 16 o C with shaking (250 rpm), harvested by centrifugation at 1100 g for 15 min, and the cellular pellet was stored at −70 o C until used.

Preparation of Whole Cell Soluble Protein Extracts
The cellular pellet of KlLeu4 and KlLeu4BIS strains was suspended in 20 mL of 50 mM K 2 HPO 4 , 0.5 M NaCl, 1 mmol L −1 EDTA, 1 mmol L −1 dithiothreitol, 1 mmol L −1 phenylmethylsulfonyl fluoride (PMSF), pH 8. Soluble extracts were obtained by sonication (Ultrasonic Processor Model: VCX130) with a tip sonicator maintaining the tubes on ice; five cycles (70% amplitude, 1 sec on and 1 sec off for 1 min) with 1 min of incubation on ice between each cycle. After centrifugation at 1100 g for 20 min at 4 o C, the supernatant was stored at 4 o C.

Immobilized Metal Affinity Chromatography (IMAC)
To purify KlLeu4 protein (KLLA0F23529, syntenic), supernatant was also purified through an equilibrated nickel column (Ni-NTA Agarose, Quiagen), and following the protocol of purification of KlLeu4 with the difference that 2 extra wash steps were added. The first extra wash was 2 volumes of 30 mmol L −1 imidazol and the second was 2 volumes of 60 mmol L −1 imidazol. To purify KlLeu4BIS protein (KLLA0D14201, non-syntenic), the supernatant was loaded on an equilibrated nickel column (Ni-NTA Agarose, Quiagen), washed with 100 volumes of lysis buffer, 50 volumes of 2 mmol L −1 imidazol, 2 volumes of 5 mmol L −1 imidazol, 2 volumes of 10 mmol L −1 imidazol and 2 volumes of 20 mmol L −1 imidazol. The protein was eluted with 2 volumes of 50, 100, 200, and 300 mmol L −1 imidazol and stored at 4 o C until used. KlLeu4 and KlLeu4BIS homogeneity was verified by denaturing with a polyacrylamide gel electrophoresis (12%, SDS-PAGE) and the gel stained with Coomassie Blue. Proteins were 10-fold concentrated with AmiconR Ultra-15 10K Centrifugal Filter Devices (Millipore), and then diluted to the original sample volume with assay buffer (50 mM K 2 HPO 4 , pH 7.5) three "washing out" cycles were performed.

α-Isopropylmalate Synthase Enzyme Assay and Protein Determination
Cells were grown to an OD 600nm ∼0.6 on glucose (2%, w/v) or ethanol (2%, v/v) as carbon sources, and samples were collected at this point. S. cerevisiae crude extracts were obtained by disrupting the cells with glass beads in phosphate buffer A (50 mM phosphate buffer pH 7.5, 1 mM PMSF and 1 mM DTT). For K. lactis strains, lysis buffer was prepared with 50 mM HEPES buffer pH 7.5 and protease inhibitor cocktail tablets (Roche, cat. No. 11697 498001). The α-isopropylmalate synthase activity was assayed with 5 mM 5,5 -dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) in 1.0 mL of medium containing 50 mM phosphate buffer pH 7.5, 140 mM KCl, 4 mM MgCl 2 , 0.25 mM acetyl coenzyme A (acetyl-CoA) and 10 mM α-ketoisovalerate (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, assay was started by adding cell free extract and the initial reaction rate was obtained from the absorbance change at 412 nm in a Varian Cary 400 spectrophotometer with a 1 cm path length. The 2-nitro-5thiobenzoic acid (NTB 2− ) production was quantified using an extinction coefficient of 14.15 M −1 cm −1 (Riddles et al., 1983). Specific activity is given in nmoles of CoA formed per minute. Protein concentration was determined as described previously (Lowry et al., 1951).

Enzyme Kinetics and Data Analysis
Initial velocity measurements were performed varying both substrates (for KlLeu4, 0.002-0.1 mM acetyl-CoA and 0.002-0.08 mM α-KIV. While for KlLeu4BIS, 0.008-0.1 mM acetyl-CoA and 0.01-0.25 mM α-KIV) and results were globally fitted to equation 1 (Eq. 1) using the Prism software. The resulting kinetic parameters are shown in Table 2.
Where  Values represent the fitted kinetic parameters ± standard error. For all the fitting analysis the R 2 was higher than 0.976. Similar Kis and Kii values were obtained using Eqs 2 or 3 as described in section "Materials and Methods" section.
Eq. 1. The resulting kinetic parameters are shown in Table 2.

Metabolite Extraction and Analysis
Cell extracts were prepared from exponentially growing cultures. Samples used for intracellular amino acid determination were treated as previously described (Quezada et al., 2008).

Data Analysis
Statistical analysis was performed using GraphPad Prism software version 8.0, for OS X. For each condition, the wild type KlLEU4 KlLEU4BIS strain was considered as control and one-way Analysis of Variance (ANOVA) tests was performed. Significant differences were evaluated by the Dunnett's multiple comparisons test. Results were considered significant at p < 0.05.
To analyze the biological function of the two paralogs genes from K. lactis, deletion mutants in each one of the two genes (Klleu4 and Klleu4bis ) were constructed (section "Materials and Methods" and Table 1). Single null mutations in either one of the two genes showed wild type growth rate in cultures supplemented with ammonium as nitrogen source and glucose (Figure 2A) or ethanol ( Figure 2B) as sole carbon sources. Only the double Klleu4 Klleu4bis mutant displayed leucine auxotrophy (Figures 2A,B). The addition of leucine to the media did not fully restore its growth rate to the wild type level (either on glucose or on ethanol) (Figures 2A,B), suggesting that simultaneous loss of KlLEU4 and KlLEU4BIS could result in an additional requirement(s) not fulfilled by the sole addition of leucine. This will be addressed below (see Discussion).
As expected, it was observed that when grown on glucose, leucine auxotrophy displayed by the Klleu4 Klleu4bis strain was complemented with plasmids harboring either one of the K. lactis paralogous genes ( Figure 3A). However, when grown on ethanol, the double mutant transformed with plasmids harboring either KlLEU4 or KlLEU4BIS, did not fully recover the wild type phenotype, not even in the presence of leucine ( Figure 3B). The K. lactis genes were cloned in a multicopy plasmid which could result in increased α-IPMS activity, and thus in a metabolic imbalance due to the draining of acetyl-CoA to α-IPM and leucine biosynthesis depleting other TCA cycle intermediates, and consequently decreasing growth rate (López et al., 2015). Although, in glucose we also see this higher activity the TCA cycle is more relevant in ethanol.
As expected, in the presence of the empty vector, leucine addition allowed growth recovery, although it did not reach wild type rate, supporting the proposal that leucine does not fulfill the need of a not yet identified, additional requirement ( Figure 3B).
Enzymatic activity of α-IPMS was determined in extracts prepared from glucose or ethanol grown cultures of the wild type strain and Klleu4 and Klleu4bis single mutants. It was found that the Klleu4 and Klleu4bis single mutant strains, retained 80 or 20% of the activity found in a wild type strain, respectively. As expected, the activity fostered by the enzymes encoded in the genes cloned in the multicopy plasmids, was higher than that found in the single mutant strains ( Figure 4A). In agreement with KlLeu4 and KlLeu4BIS catalytic contribution, the leucine intracellular pools were higher in the Klleu4 mutant as compared to that observed in the Klleu4bis mutant ( Figure 4B).
Previous results showed that ScLEU4 expression is influenced by the nature of the carbon source (López et al., 2015). Thus, we analyzed whether a similar effect could be observed for KlLEU4 and KlLEU4BIS. Therefore, total RNA preparations were obtained from wild type K. lactis glucose or ethanol-grown cultures. Figure 4C shows that both genes have similar expression on either carbon source. We also analyzed the expression profile of each gene when its paralogous was deleted. It was found that absence of either one of the two paralogous genes did not affect expression of the remaining one ( Figure 4C).

K. lactis Isozymes Are Located in Different Subcellular Compartments
The phylogeny of the α-IPMS encoding-genes, discussed below and in Supplementary Material, strongly suggested the KlLeu4 paralog to be mitochondrial, and KlLeu4BIS to be cytosolic. Figure 5A shows this the case. Thus, it is extremely unlikely that hetero-oligomeric isoforms could form "in vivo, " suggesting that the enzymatic activity detected in a wild type strain results from the addition of the independent activity of the KlLeu4 and KlLeu4BIS homodimeric isozymes.

KlLeu4 and KlLeu4BIS Are Leucine Sensitive
Leucine sensitivity of the two α-isopropylmalate synthases paralogs of K. lactis was analyzed. Half-maximal inhibitory concentration (IC 50 ) of KlLeu4 and KlLeu4BIS was determined in whole cell extracts obtained from either Klleu4 or Klleu4bis single mutants. As Figure 5B shows, the two K. lactis paralogs isozymes showed similar leucine sensitivity: KlLeu4 0.020 mM and KlLeu4BIS 0.037 mM.
The kinetic properties of the purified enzymes (see section "Materials and Methods") were analyzed (Supplementary Figure S1). Experimental data were fitted to Eq. 1, which corresponds to a compulsory ordered bi-bi reaction under steady state treatment (Segel, 1993). The kinetic properties of the enzymes were similar ( Table 2). For both enzymes leucine behaved as a mixed inhibitor, decreasing both, the apparent affinity for the substrates and the apparent maximum enzyme reaction rate (Vmax). Experimental data were fitted to Eq. 2 when the acetyl-CoA concentration was varied and to Eq. 3 when the variable substrate was α-KIV (Segel, 1993) (Table 2) (see text and equations in section "Materials and Methods"). This model predicts that leucine can bind for both isoforms either to the free enzyme or to the substrate-bound enzyme. The former process is reflected in the Kis constant whereas the latter is reflected in the Kii value (Table 2, Eqs 2 and 3 and Figure 5B). From these kinetic parameters we can thus conclude that both enzymes contribute very similarly to the control of the metabolic flux, at variance to the situation in S. cerevisiae in which the Leu9 homomeric enzyme showed a Kii value which was 24-and 20-fold higher than the one found for the Leu4 homodimer and the Leu4-Leu9 heterodimer, respectively.
As single mutants showed wild type growth phenotypes (see above, Figure 2), KlLeu4 and KlLeu4BIS, although located in different subcellular compartments, must have a redundant role in leucine biosynthesis under the conditions tested.
Heterologous Complementation of S. cerevisiae Double (Scleu4 Scleu9 ) and Single (Scleu4 ScLEU9) Mutants Subcellular localization of the K. lactis orthologous isozymes heterologously expressed in S. cerevisiae was examined. As shown in Figure 6A, in S cerevisiae, both enzymes maintained their native localization: KlLeu4-mitochondrial and KlLeu4BIScytosolic. In an Scleu4 ScLEU9 strain transformed with the KlLeu4 mitochondrial isoform, KlLeu4-ScLeu9 heterodimers could presumably be formed, thus decreasing ScLeu9-ScLeu9 homodimer concentration, hence counteracting the negative effect on growth of the ScLeu9-ScLeu9 leucine resistant isoform.
Double Scleu4 Scleu9 or single Scleu4 ScLEU9 or mutants were independently transformed with YEpKD352 plasmids each harboring KlLEU4 or KlLEU4BIS with their  In (A,B), bars indicate mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. (C) Northern analysis was carried out as described in section "Materials and Methods." Total Yeast RNA was prepared from 100 mL aliquots of cultures grown to an OD 600nm ∼0.6 on minimal media with 2% w/v glucose or 2% v/v ethanol as carbon sources. Specific probes are described in section "Materials and Methods." Blots were scanned using the program ImageQuant 5.2 (Molecular Dynamics). endogenous promoters. As controls, S. cerevisiae mutants were also transformed with ScLEU4 and ScLEU9 cloned in YEpKD352. We found that, as previously reported (López et al., 2015), when grown on glucose or ethanol, Scleu4 Scleu9 transformed with ScLEU4 grew as well as the wild type strain. However, double mutants transformed with ScLEU9 were only partially complemented in glucose and not complemented in ethanol (Figures 7A,B). The Scleu4 ScLEU9 single mutant strain, when transformed with ScLEU4 recovered a wild type phenotype, while transformants obtained with ScLEU9 showed a similar phenotype to that displayed when transformed with the empty YEpKD352 vector (Figures 7C,D). A vector carrying KlLEU4 partially complemented the phenotype of an Scleu4 ScLEU9 strain, while one carrying KlLEU4BIS did not complement at all (Figures 7C,D).
Leucine pools were determined in the wild type strain (ScLEU4 ScLEU9), Scleu4 ScLEU9, and Scleu4 LEU9 transformed with YEpKD352 empty vector or YEpKD352-ScLEU4, ScLEU9, KlLEU4, or KlLEU4BIS. As previously reported, the Scleu4 ScLEU9 mutant accumulated a several-fold higher leucine pool, in either glucose or ethanol, as compared to that found in the wild type strain (Figure 6B). This accumulation was reverted in ScLEU4 transformants, but not in ScLEU9 transformants (López et al., 2015; Figure 6B). Surprisingly, although KlLEU4 but not KlLEU4BIS improved the growth rate of the Scleu4 ScLEU9 strain, both KlLEU4 and KlLEU4BIS transformants showed intermediate leucine accumulation, ranging between the one observed in the Scleu4 ScLEU9 mutant and that found in the Scleu4 ScLEU9 transformed with ScLEU4 ( Figure 6B). In an Scleu4 ScLEU9 strain, LEU9 expression is several-fold enhanced ( Figure 6C) this together with the leucine insensitivity of ScLeu9-ScLeu9 homodimers would account for the increased leucine pool (López et al., 2015).

Scleu4 ScLEU9-Dependent Induced
ScLEU9 Over-Expression Is Suppressed in Transformants Harboring ScLEU4, KlLEU4, or KlLEU4BIS Northern blot analysis showed that the intermediate leucine pools observed in the Scleu4 ScLEU9, transformed with YEpKD352-ScLEU4, YEpKD352-KlLEU4, or YEpKD352-KlLEU4BIS could be attributed to diminished ScLEU9 expression, as compared to that found in the Scleu4 ScLEU9/YEpKD352 (Figure 6C). Both the K. lactis genes mimic the ScLEU4 repressive effect on ScLEU9 expression by hitherto unidentified mechanisms. In a Scleu4 ScLEU9 mutant, the reduced leucine sensitivity of ScLeu9-ScLeu9 homodimers would result in increased flux through the leucine biosynthetic pathway, depleting the pool of metabolic intermediates, leading to reduced growth. In the Scleu4 ScLEU9/KlLEU4 transformed strain, ScLEU9 expression is decreased, and the reduced growth phenotype is partially alleviated. Additionally, the possible formation of a KlLeu4-ScLeu9 leucine sensitive heterodimer could also contribute to the alleviated growth phenotype. Transformants obtained with KlLEU4BIS, encoding the cytosolic isoform, which is presumably unable to form heterodimers with ScLEU9, are not Leucine pools of wild type ScLEU4 ScLEU9 and derived single ScLEU4 mutant harboring plasmids YEpKD352-ScLEU4, YEpKD352-ScLEU9, YEpKD352-KlLEU4, or YEpKD352-KlLEU4BIS were prepared as described in section "Materials and Methods. Bars indicate mean ± SD (n = 3), * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001. (C) Northern analysis was carried out as described in section "Materials and Methods." Total Yeast RNA was prepared from 100 mL aliquots of cultures grown to an OD 600nm ∼0.6 on minimal media with 2% w/v glucose as carbon source. Probes used to monitor expression for each gene are described in section "Materials and Methods." Blots were scanned using the program ImageQuant 5.2 (Molecular Dynamics). complemented for the impaired growth phenotype although ScLEU9 expression is diminished (Figures 6C, 7C,D).

Phylogeny of α-Isopropyl Malate Synthase in the Saccharomycotina
Isopropyl malate synthase (α-IPMS) is conserved throughout the tree of life (with the notable exception of metazoans) and it is present in most fungal taxa. The two mitochondrial paralogs of S. cerevisiae analyzed in our previous publication (López et al., 2015) originated from the WGD event, which is at the root of the sub-family of Saccharomycetaceae that includes S. cerevisiae (Wolfe and Shields, 1997;Marcet-Houben and Gabaldón, 2015;see below).
In this paper, we describe two paralogous K. lactis genes encoding leucine sensitive α-IPMSs which differ in their cellular localization. The genes encoding these two isoforms must have originated by a single gene duplication preceding the WGD. We have attempted to pinpoint the origin of this duplication by constructing a phylogeny of α-IPMSs within the  In these clades only one paralog is extant, presumably cytosolic (see text and Supplementary Data Sheet 1). The blue triangle includes basal species of Saccharomycotina where no α-IPMS gene duplication has occurred. Within the Saccharomycotina, a gene duplication occurred (green and brown triangles), leading to the genomes of most species comprising two isoforms, presumably cytosolic and mitochondrial respectively. KlLEU4BIS and KlLEU4 are included in the green and brown clade respectively. In several pre-WGD members of the Saccharomycetacea (represented by a yellow triangle), including those giving origin to the WGD, the cytosolic paralog was lost. Thus, the WGD results in two mitochondrial isoforms (red triangle). ScLEU4 and ScLEU9 originate from this duplication. Figure S2). The WGD arose seemingly from the hybridization of two distinct species of Saccharomycetaceae, one belonging to the KLE (Kluyveromyces, Lachancea, and Eremothecium) clade, the other to the ZT (Zygosaccharomyces and Torulaspora) clade (Marcet-Houben and Gabaldón, 2015). In both clades, episodes of loss of the cytosolic paralog have occurred (Supplementary Figure S2, noticeably in Lachancea species and T. delbrueckii), thus resulting in post-WGD species including two mitochondrial isoforms but no cytoplasmic isoforms, not withstanding that further loss of one of the two mitochondrial paralogs has occurred in a few post-WGD species (such as S. pastorianus, see Supplementary Data Sheet 1 for details).

DISCUSSION
This study addresses the question of whether paralogous genes with divergent evolutionary origin constituting orthologous pairs, such as the ScLEU4/ScLEU9 vs. KlLEU4/KlLEU4BIS, follow similar or diverse functional diversification patterns.
Previous work established that putative paralogs, arising from segmental or single gene duplications are extant in species of Saccharomycetales upstream the WGD episode (Langkjaer et al., 2003;Dujon et al., 2004). Taking advantage of the MetaPhOrs database 7 (Pryszcz et al., 2011;Chorostecki et al., 2020), we have estimated that in K. lactis 162 gene duplications have occurred since its divergence from the basal species Yarrowia lipolytica. Incidentally, some duplicated pairs encode proteins involved in amino acid biosynthesis, such as homologs of the post WGD pairs the S. cerevisiae SER3/SER33 and LYS20/LYS21. After the α-IPMS duplication event, which originated the K. lactis KlLEU4/KlLEU4BIS pair, episodes of loss occurred in some clades, including within the pre-WGD Saccharomyceteacea. Some species lost the cytosolic paralog. In others, through loss of the mitochondrial pre-sequence, the paralog arising from the original duplication reverted to a presumably cytosolic location. In this work we show that synthesis of α-IPMS could occur either in the cytosol or the mitochondria. Perhaps the successive changes of localization of the α-IPMS are related to the specific ecology and/or physiology of each species.
ScLEU4 and ScLEU9 encoded isozymes, are both mitochondrially located; however, a fraction of ScLeu4 resides in the cytosol and could thus synthesize α-IPM in this location. Accordingly, an oac1 mutant, which is unable to transport α-IPM from the mitochondria to the cytosol, is able to sustain a braditrophic growth feeding from the leucine synthesized in the cytosol (Marobbio et al., 2008). We have previously shown (López et al., 2015) that ScLEU4 and ScLEU9 originated from the WGD event, and have functionally diverged, resulting in three mitochondrial isoforms: the leucine-sensitive homodimeric ScLeu4-ScLeu4, the relatively leucine-resistant homodimeric ScLeu9-ScLeu9, and the heterodimeric ScLeu4-ScLeu9 displaying intermediate leucine sensitivity (López et al., 2015).
Our results indicate that K. lactis KlLEU4 and KlEU4BISencoded isozymes, which originated from a more ancient small-scale duplication event (SSD), are both leucine sensitive isozymes which, under the conditions of the present study, play a redundant role, since both genes can complement the wild type phenotype to the Klleu4 Klleu4bis leucine auxotrophic mutant. Divergence has been observed in their subcellular localization, KlLeu4 being mitochondrial and KlLeu4BIS cytosolic. We have asked whether each of the K. lactis orthologous pair (KlLEU4/KlLEU4BIS) could complement the complex phenotype of a Scleu4 ScLEU9 mutant and the leucine auxotrophy of the Scleu4 Scleu9 double mutant. Our results indicate that either K. lactis paralog can complement the leucine auxotrophy. However, the complex Scleu4 ScLEU9 phenotype is only complemented by the KlLEU4 gene which is syntenic with ScLEU4 and ScLEU9 and encodes the mitochondrial isoform.
Is the Function of the K. lactis KlLEU4 and KlLEU4BIS Paralogous Genes Redundant?
The growth phenotype of the double Klleu4 Klleu4bis mutant is not restored to wild type levels by the sole addition of leucine, suggesting the existence of an additional requirement not supplemented by this amino acid. The transcription factor ScLeu3 activating function is positively modulated by α-IPM (Sze et al., 1993;Boer et al., 2005). ScLeu3 ligated to α-IPM activates transcription of various genes involved in branched chain amino acid and glutamate biosynthesis (Hu et al., 1995;Kohlhaw, 2003). Absence of α-IPM production in the Klleu4 Klleu4bis double mutant could alter the presumed transcriptional role carried out by the KlLEU3 orthologous gene (KLLA0D10593g, NCBI Gene ID 2892795, ORF 52% identity with ScLEU3). It is not far-fetched to assume that the K. lactis ortholog also requires α-IPM-mediated activation. The partial, rather than complete, supplementation by leucine of the double Klleu4 Klleu4bis mutant, as opposed to that found for the Scleu4 Scleu9 strict leucine auxotroph, could be due to an α-IPM requirement, not satisfied by leucine. Although KlLEU4 and KlLEU4BIS seem to play redundant roles; it may be that in some physiological conditions the simultaneous activity of both enzymes could be necessary to attain physiological α -IPM levels.
K. lactis is a strict aerobic organism, and its respiratory system is not glucose repressed (Schaffrath and Breunig, 2000), Klleu4 and Klleu4bis single mutants show wild type growth rate on either glucose or ethanol as carbon and energy sources, indicating that each paralogous protein is capable to maintain energy and biomass production in aerobiosis.
The physiological function of the different localization of the first step of the pathway in S. cerevisiae and K. lactis are not obvious. KlLEU4 and KlLEU4BIS seem to play redundant roles; however, the fact that the encoded enzymes show a differential subcellular localization, which has been maintained in several families of the Saccharomycetales (see Supplementary  Material) is somewhat of a paradox. Further studies will have to be carried out in order to determine whether these genes could play non-redundant roles and their relation to α-IPM and leucine biosynthesis.

The Complex Scleu4 ScLEU9
Phenotype Can Be Complemented Only by the KlLeu4 Mitochondrial Isozyme KlLEU4 and KlLEU4BIS encoding respectively, mitochondrial and cytosolic isoforms, are able to fully complement the leucine auxotrophy displayed by Scleu4 Scleu9 double mutant.
We have previously shown that over-expression of ScLEU9 in a Scleu4 genetic background, results in slow growth (on either glucose or ethanol as carbon sources) and leucine accumulation. ScLeu9-ScLeu9 resistance to leucine feedback inhibition could impact flux control due to increased activity of the biosynthetic pathway (López et al., 2015). Accordingly, when the Scleu4 ScLEU9 mutant is complemented with the ScLEU4 the heterodimeric leucine-sensitive ScLeu4-ScLeu9 isozyme is formed, thus recovering the physiological feedback regulation, wild type leucine biosynthetic rate and consequently wild type growth. The KlLeu4 mitochondrial isoform is able to only partially complement the growth phenotype of the mutant Scleu4 ScLEU9, on either glucose or ethanol, partially mirroring ScLeu4 activity, while the cytosolic KlLeu4BIS isozyme did not improve Scleu4 LEU9 growth phenotype either on glucose or ethanol. Both K. lactis paralogs can revert the increased ScLEU9 expression to wild type levels ("Results" sections, Figure 6C). Thus, Scleu4 ScLEU9 growth rate complementation by the KlLEU4 orthologous gene could not only be due to the KlLEU4 capacity to counteract the enhanced ScLEU9 expression in a Scleu4 background. Additionally, the mitochondrial localization of the KlLeu4 protein could in principle enable the formation of a heterodimeric ScLeu9-KlLeu4 isoform, which may have higher leucine sensitivity than the ScLeu9-ScLeu9 homodimer, thus overcoming decreased feedback regulation. While KlLeu4BIS can prevent enhanced ScLEU9 expression, its cytosolic localization would not allow the formation of the proposed ScLeu9-KlLeu4BIS leucine sensitive heterodimers.

CONCLUSION
It can be concluded that the KlLEU4 and KlLEU4BIS paralogous pair have followed a path of functional diversification resulting in different subcellular localization of the encoded enzymes, which at variance from that seen in S. cerevisiae, do not differ in leucine sensitivity thus not affecting differentially α-IPMS biosynthesis feedback regulation.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/Supplementary Material.  (http://dgapa.unam.mx), Consejo Nacional de Ciencia y Tecnología grant CB-2014-239492-B, grant from the Agencia Mexicana de Cooperación Internacional Para el Desarrollo Cooperación Científica y Tecnológica México-Italia 2015-2017, and grant NoCRP/MEX10-03 from the International Centre for Genetic Engineering and Biotechnology. ML had a CONACYT Master degree fellowship. XE-F was supported with a postdoctoral grant from CONACYT (CVU 420248). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.