Hexose Transport in Torulaspora Delbrueckii: Identification of Igt1, A

FEMS Yeast Research, 20, 2020, foaa004

doi: 10.1093/femsyr/foaa004 Advance Access Publication Date: 25 January 2020 Research Article Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020

RESEARCH ARTICLE Hexose transport in Torulaspora delbrueckii: identification of Igt1, a new dual-affinity transporter Andreia Pacheco1, Lorena Donzella1,2, Maria Jose Hernandez-Lopez3,Maria Judite Almeida1, Jose Antonio Prieto3, Francisca Randez-Gil3, John P. Morrissey2,† and Maria Joao˜ Sousa1,*

1Centre of Environmental and Molecular Biology, Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal, 2School of Microbiology, Centre for Synthetic Biology and Biotechnology, Environmental Research Institute, APC Microbiome Institute, University College Cork, T12YT20 Cork, Ireland and 3Department of Biotechnology, Instituto de Agroqumica y Tecnologia de los Alimentos, Consejo Superior de Investigaciones Cientficas, Avda. Agustn Escardino, 7. 46980-Paterna, Valencia, Spain

∗Corresponding author: Centre of Environmental and Molecular Biology, Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal. Tel: +351253601545; E-mail: [email protected] One sentence summary: Here the authors identified and characterized, both from a structural and a biochemical point of view, a new intermediate affinity hexose transporter, Igt1, in the industrial relevant yeast T. delbrueckii. Editor: Carol Munro †John P. Morrissey, http://orcid.org/0000-0001-7960-2001

ABSTRACT Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomyces cerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured byKm, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic glucose uptake kinetics with an intermediate- (Km = 6.5 ± 2.0 mM) and a high-affinity (Km = 0.10 ± 0.01 mM) component, whereas cells grown in 2% glucose displayed monophasic kinetics with an intermediate-affinity (Km of 11.5 ± 1.5 mM). This work contributes to a better characterization of glucose transport in T. delbrueckii, with relevant implications for its exploitation in the food industry.

Keywords: Torulaspora delbrueckii; Glucose transport; hexose transporter; transport kinetics; dual affinity; glucose transporter gene cluster

INTRODUCTION et al. 1997) that is of increasing industrial interest. It has a positive effect on the taste and aroma of alcoholic bev- Torulaspora delbrueckii is a yeast phylogenetically related to erages, such as wine and beer, exhibiting low production of Saccharomyces cerevisiae (James, Collins and Roberts 1996;Oda undesired metabolites and increased levels of flavor-active

Received: 8 December 2019; Accepted: 24 January 2020

1 2 FEMS Yeast Research, 2020, Vol. 20, No. 1

positive-contributing compounds (Cabrera et al. 1988;Martinez YPD media (1%, w/v, yeast extract, 2%, w/v, peptone and 2%, et al. 1990; Ciani and Ferraro 1998). In addition, the use of T. de l - w/v, glucose) or SD (0.67%, w/v, yeast nitrogen base without brueckii under standard conditions, in mixed or sequential cul- amino acids, DIFCO) plus glucose or maltose at concentrations ture with S. cerevisiae, has been proposed as a way of reducing indicated in results and supplemented with the appropriate the acetic acid content in wine (Ciani and Picciotti 1995;Ciani, auxotrophic requirements (Sherman 1991). Yeast transformants Beco and Comitini 2006). Some T. delbrueckii strains are also containing the geneticin resistance module (kanMX4) were frequently found among isolates from traditional corn and rye selected on YPD-agar plates supplemented with 300 μg/L of G418 bread doughs from northern Portugal (Almeida and Pais 1996a). (geneticin, Sigma-Aldrich), added after autoclaving and cooling Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020 These strains exhibit a very good baking ability and exceptional to 60◦C. LB medium was prepared as previously described (Sam- resistance to osmotic (Hernandez-Lopez, Prieto and Randez- brook, Maniatis and Fritsch 1989). When necessary 100 μg/mL of Gil 2003) and freeze-thaw stresses (Almeida and Pais 1996b), ampicillin was added to standard LB plates or liquid after auto- opening the possibility of their application in high-sugar and claving and cooling to 60◦C. A sterile filtered stock solution was frozen-dough fermentations. In spite of the interest on this yeast used in this case. species, there is still limited information about relevant aspects of its biology and biochemistry, including on the transport mech- Reagents anisms and transporters repertoire assuring the uptake of sugars during fermentation, a limiting step in the metabolism of Oligonucleotides were purchased from MWG Biotech, Germany. carbon compounds during fermentation both in S. cerevisiae Restriction and modification enzymes were from Roche Applied and T. delbrueckii (Diderich et al. 1999;Yeet al. 1999); (Alves- Science, Germany. Accuzyme DNA Polymerase was obtained Araujo´ et al. 2007). In S. cerevisiae, the uptake of hexoses occurs from Bioline, Germany. through facilitated diffusion (Lagunas 1993) mediated by several transporters, the Hxt proteins. These proteins are members of CHEF- pulsed field gel electrophoresis (PFGE) the major facilitator superfamily (MFS), a group of membrane- bound transport proteins that share a common ancestral origin Intact chromosomal DNA for PFGE was prepared in plugs as (Andre 1995). Proteins belonging to the MSF family exhibit strong previously described (Ribeiro, Corte-Real and Johansson 2006). structural conservation (Vardy et al. 2004), usually consisting of a PFGE was run in a CHEF-DRII Chiller System (Bio-Rad, Hercules, single integral membrane protein with two sets of six hydropho- CA). PFGE gels were run in 0.5% (w/v) Tris borate-EDTA buffer at ◦ ◦ bic transmembrane-spanning (TMS) α-helices connected by a 12 Cwithanangleof120 with the following voltage and switch hydrophilic loop. The S. cerevisiae Hxt family includes 20 differ- times: 480s → 900s, 3 v/cm for 10 hours; 240s → 480s, 3 v/cm ent hexose transporter related proteins, Hxt1p–Hxt17p, Gal2p, for 15 hours; 120s → 240 s, 3 v/cm for 15 hours; 90 s, 6 v/cm for Snf3p and Rgt2p. Several other hexose transporters have also 10 hours and 60 s, 6 v/cm for 5 hours. Gels were subsequently been identified in non-Saccharomyces yeasts like Kluyveromyces stained with 0.8% (w/v) ethidium bromide solution for 45 min lactis, Kluyveromyces marxianus, Schizosaccharomyces pombe, Pichia and destained for 20 min. Gels were visualized under UV light stipites, Yarrowia lipolytica, Candida glycerinogenes and Candida albi- and analyzed using the EagleEye II Image Acquisition System cans with different kinetic properties and modes of regulation (Stratagene, La Jolla, CA). Afterwards CHEF-PFGE was blotted on (Leandro, Fonseca and Goncalves 2009; Lazar et al. 2017;Liang to a positively charged nylon membrane and the membrane was et al. 2018; Varela et al. 2019). We have previously shown that hybridized with a 467 bp length probe homologous to LGT1 and T. delbrueckii PYCC 5321 displays a mediated glucose transport IGT1 C-terminus. activity best fitted assuming a biphasic Michaelis–Menten kinetics with a low and a high-affinity component. However, until Construction of LGT1 disrupted and overexpressing now, just one glucose transporter has been characterized in strains this yeast, the low-affinity glucose transporter Lgt1, that can also mediate the uptake of fructose (Alves-Araujo´ et al. 2005). Standard DNA manipulations were carried out as previously These results suggested the occurrence of other functional hex- described (Sambrook, Maniatis and Fritsch 1989). The LGT1 dis- ose transporters in T. delbrueckii. ruption cassette was obtained by restriction followed by sub- In this manuscript, we report the identification and charac- cloning as described in Pacheco, Almeida and Sousa (2009). terization of a second glucose transporter from this yeast, Igt1. The PvuII-loxP-KanMX-loxP-SpeI fragment released by restriction Kinetic analysis of sugar transport mediated by Igt1 in a hex- from plasmid pUG6 (Goldstein and McCusker 1999) was cloned ose transport-null mutant strain of S. cerevisiae, showed that this into BalI/NheI restricted YepHxt6 (a plasmid containing the LGT1 transporter mediates uptake of glucose with intermediate and open reading frame (ORF) and part of the gene promoter and ter- high affinity, depending on the concentration of the sugar in minator regions) (Alves-Araujo´ et al. 2005), creating YLgt1kan. the medium. BLAST analyses of the reference genome of T. de l - The LGT1 disruption cassette, which contains the loxP-KanMX- brueckii CBS 1146 with LGT1 and IGT1, showed that they are prob- loxP module flanked by 320 and 345 bp (5 and 3 sides, respec- ably part of a larger cluster of hexose transporters. Despite the tively), homologous to each margin of LGT1,wasgeneratedby different kinetic behavior of the two transporters, Lgt1 and Igt1 PCR using the YLgt1kan plasmid as template. Correct disruption display a high degree of sequence similarity. of the LGT1 gene was detected by diagnostic PCR using whole yeast cells (Huxley, Green and Dunham 1990) from isolated colonies and a set of oligonucleotides designed to bind outside MATERIALS AND METHODS or inside of the replaced LGT1 sequence and within the marker Strains, media and growth conditions module. To obtain a T. delbrueckii LGT1 overexpressing strain, a 1750 bp XmaI-LGT1-XhoI 1750 fragment containing the LGT1 ORF Torulaspora delbrueckii PYCC 5321, and a glucose transport-null was PCR-amplified from genomic DNA of T. delbrueckii PYCC 5321 mutant of S. cerevisiae, EBY.VW4000 (Wieczorke et al. 1999)were andclonedintotheXmaI/XhoI restricted and dephosphorylated used throughout this work. Yeast cells were cultured at 30◦Cin p426GPD vector (Mumberg, Muller and Funk 1995), creating the Pacheco et al. 3

pGPDLGT1 vector. The oligonucleotides used were: 0:sc GCGCC when the regression was best fitted to two equations, values CGGGATGTCTACTACAGA/0:sc and GCG0:underline CTCGAG were obtained from Eadie–Hofstee plots. /0:underlineTTATTTGGAGAAAA (the added XmaI/XhoIrestric- tion sites for cloning are underlined). Phylogenetic analysis and promoter characterization The 2384 bp SacI-GPD prom-Lgt1-XhoI fragmet, from pGPDLGT1was cloned on pRS41K centromeric plasmid vector, Analysis of sequence data was carried out using DNAMAN creating pGPDK. Afterwards the 262 bp XhoI-CYC term–KpnI sequence analysis software (Lynnon Biosoft). Similarity searches fragment from pGPDLGT1 was cloned into pGPDK creating were performed at the MCBI database using BLAST software Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020 pGPDLGT1K, which contains the LGT1 gene under the regu- (Altschul et al. 1997). Neighbor-joining phylogenetic analysis lation of the S. cerevisiae GPD1 promoter from p426GPD. DNA and tree building were performed using MEGA6 (Tamura et al. fragments resolved in agarose gels were purified by use of a 2007). Protein sequence alignment was obtained using MUS- QIAquick gel extraction kit (Qiagen GmbH, Hilden, Germany). CLE 3.8 (Edgar 2004) and visualized by Jalview v.2 (Waterhouse et al. 2009). Synteny conservation was inspected using the Yeast Sub-cloning of IGT1 gene Genome Order Browser (YGOB, www.ygob.ucd.ie) (Byrne and Wolfe 2005). Plasmid YEpIGT1 was generated by cloning a 2760 bp PstI–EcoRI IGT1 and LGT1 promoter characterization was carried out fragment from plasmid YEpT2 (Alves-Araujo´ et al. 2005), contain- using the YeTFaSCo database (de Boer and Hughes 2012)toiden- ing the whole IGT1 gene, 590 bp of the promoter and 497 bp tify potential transcription factor binding sites. After filtering out of the terminator into the PstI–EcoRI sites of the YEplac181 hits with low identity (<85%), the resulting binding sequences vector (Gietz and Sugino 1988). The oligonucleotides used were plotted against the sequence of the IGT1 or LGT1 promoter were: CGC0:underline CTGCAG/0:underlineTTGTCCAGACAG and visualized with SnapGene software (from GSL Biotech; avail- CACC and CGC0:underline GAATTC/0:underlineCCATCTTC able at snapgene.com). CGCCAAGC (PstI–EcoRI restriction sites underlined). 3-D structure prediction Yeast transformation Protein structure and function prediction were performed All yeast transformations were performed using the lithium using the I-TASSER online platform from the Yang Zhang’s acetate protocol as previously described (Schiestl and Gietz Research Group (https://zhanglab.ccmb.med.umich.edu/I-TASS 1989). Correct yeast transformations were verified, by plasmid ER/)(Eswaret al. 2008) and figures were prepared with PyMOL DNA isolation using the ChargesSwitch plasmid yeast mini kit v.2.3 (DeLano 2002). The FASTA file containing the amino-acid (Invitrogen, USA) and subsequent transformation in Escherichia sequence for Igt1 was obtained from the GenBank database coli according to SEM method (Inoue, Nojima and Okayama (available at http://www.ncbi.nlm.nih.gov/GenBank/index.html) 1990). under accession number [FJ899142] and submitted to the I- TASSER online server. The structure prediction consists in the Glucose uptake assays alignment of the target sequence to a protein template, which is identified by LOMETS, a method that selects the top ten align- For glucose uptake assays, [U-14C] glucose with a specific activ- ments in a PDB library. The full-length structure models are ity of 310 mCi/mmol (Amersham) was used. The cells were har- constructed by an interactive fragment assembly simulation, in vested at exponential phase (OD640nm = 0.5–0.6) by centrifuga- which the unaligned regions of the sequence are built through tion, washed twice with cold water and suspended in water to ab initio folding, by considering the lowest free energy states a cellular density of 35–45 mg (biomass dry weight)/mL. Glucose and minimized steric clashes. The final models are built at an uptake assays were performed as described earlier (Alves-Araujo´ atomic level by REMO, which optimizes the hydrogen-bonding et al. 2005). To estimate labeled glucose uptake rates, 10 μLof profile. In addition, the I-TASSER online software also allows the the yeast suspension, kept on ice, was mixed with 30 μLof0.1M prediction of protein biological functions through the compar- KH2PO4 buffer, pH 5.0, and the mixture was allowed to warm at ison with known proteins in the function database (Yang and ◦ 30 C. The reaction was started by addition of 10 μL of an aque- Zhang 2015a; Yang and Zhang 2015b). The predicted sequence ous solution of [U-14C] glucose to the final desired concentra- of Igt1 was submitted to the TMHMM 2.0 server (Sonnham- tion from 0.1–100 mM and stopped, after incubation for 5 s, by mer, von Heijne and Krogh 1998), SOSUI (Hirokawa, Boon- dilution with 5 mL of chilled water. The cells were immediately Chieng and Mitaku 1998) and TMPRED (Stoffel 1993), POLYVIEW collected on glass fiber filters (Whatman, Clifton, NJ, USA) and (Porollo, Adamczak and Meller 2004) and PHOBIUS (Kall, Krogh washed with 10 mL of chilled water. Non-specific binding of [U- and Sonnhammer 2004) for the prediction of transmembrane 14C] glucose to the yeast cells was determined by pouring ice- domains. cold water immediately before the addition of the labeled glucose. The filters were immersed and radioactivity was measured with a liquid scintillation counter (Packard Instrument Co., Inc.). RESULTS To determine the inhibiting effect of other sugars on glucose Torulaspora delbrueckii has other(s) transport, competition experiments were carried out by mea- physiological-relevant glucose transporters besides suring the uptake of glucose at each concentration in the pres- LGT1 ence of an excess of the other unlabeled sugar. The two sugars were added simultaneously to the reaction mixture. The concen- We previously isolated and characterized LGT1, coding for the tration of the inhibitors was 100 mM. Triplicate determinations first identified hexose transporter from T. delbrueckii. To further were performed at each glucose concentration. Kinetic param- investigate the impact of Lgt1-mediated transport in overall glu- eters were derived using computer-assisted non-linear regres- cose transport of T. delbrueckii, we created a lgt1 mutant by sion using GraphPad Prism software (Microsoft Corp.); however, targeted gene disruption. Deletion of this hexose transporter 4 FEMS Yeast Research, 2020, Vol. 20, No. 1 Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020

Figure 1. Glucose transport in T. delbrueckii cells deficient in Lgt1 (A), Eadie-Hofstee plot of glucose initial uptake rates in cells of T. delbrueckii PYCC 5321 () and lgt1 (). (B), Kinetic parameters of the low-affinity component of glucose transport determined for Wtand lgt1 strains. Vmax is indicated by the y-intercept, while the slope of the line reflects the Km. Two-way ANOVA analysis revealed that differences between strains were extremely significantP ( < 0.0001). The cells were grown in ◦ YPD 2% glucose (w/v), at 30 C and harvested at exponential phase (OD640 = 0.5–0.6).

gene resulted in a small decrease of glucose uptake rates, only evident at the higher concentration range. The apparent Km of the low-affinity component did not change butmax theV decreased by approximately 20% (Fig. 1). These results are consistent with the low-affinity of Lgt1 for glucose and point tothe existence of other physiologically relevant hexose transporters in this yeast. Also in agreement with these results, the lgt1 null strain did not present any growth defect in glucose media (not shown). The same has been described for individual hxt mutants in S. cerevisiae, which did not show any growth phenotype, nor dramatic changes in uptake kinetics (Reifenberger, Freidel and Ciriacy 1995). Additionally, we constructed a plasmid to over- express LGT1 and confirmed its functionality for glucose transport using the S. cerevisiae hxt-null mutant strain EBY.VW4000. Torulaspora delbrueckii strains transformed with this plasmid did not display any changes in glucose uptake rates, suggesting that expression of the gene is not limiting glucose transport (data not shown).

IGT1, a putative new glucose transporter gene arranged in a cluster with LGT1

Since our results above indicated that there were other relevant glucose transporters in T. delbrueckii,wewentontoiden- tify and characterize further potential candidates. In previous work (Alves-Araujo´ et al. 2005), using a genomic library of T. de l - brueckii PYCC 5321, we had selected four S. cerevisiae EBY.VW4000 (hxt null strain) transformants, able to grow on glucose as a Figure 2. IGT1 and LGT1 chromosome distribution. lane1: T. delbrueckii PYCC 5321 sole carbon source. DNA sequencing of the plasmid insert from CHEF- PFGE membrane hybridization with a probe homologous to LGT1 and IGT1 one of these transformants, YEpT2, revealed the presence of a C- terminus. lane 2: T. delbrueckii PYCC 5321 and lane 3: S. cerevisiae karyotype. 1707 bp uninterrupted ORF showing homology (70%–80% iden- For a reference, the size of some of the S. cerevisiae chromosomes is indicated. tity) to known yeast hexose transporters and 70% total identity with LGT1. We named the new ORF IGT1, for intermediate glucose transporter (see below). Downstream of this ORF, we also neither of these genes are to be found in additional copies on found part of the LGT1 promotor indicating that the two genes another chromosome in this strain. are arranged in tandem. Looking at the T. delbrueckii reference genome from CBS 1146 Hybridization of PFGE-CHEF with a probe homologous to a strain (Gordon et al. 2011), we found in Chromosome 5 the conserved region (C terminal region) of LGT1 and of the IGT1 presence of a cluster with 3 genes annotated as putative Hxt was used to gain information on the location of the two genes orthologs (TDEL 0E02310; TDEL 0E02300 and TDEL 0E02280) and or the existence of other possible copies on the genome of T. de l - a fourth gene (TDEL 0E02290) that also displays high identity to brueckii PYCC 5321. The results in Fig. 2 show that the probe only HXT genes (Fig. 3A). TDEL 0E02310 and TDEL 0E02300 are 99% hybridized with one chromosome, which is in accordance with identical to LGT1 and IGT1, respectively, confirming that these the tandem arrangement of LGT1 and IGT1, and establishes that are orthologous genes. Since, we found that LGT1 and IGT1 are Pacheco et al. 5 Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020

Figure 3. IGT1 and LGT1 gene cluster evolutionary relationships. (A), Schematic illustration for the IGT1 and LGT1 gene cluster syntenic structure and evolution prediction. (B), Neighbor-joining phylogenetic analysis and compact tree building were performed using MEGA6/MUSCLE, comparing Igt1 (ACX37503.1) and Lgt1 (AAT95983.1) of Torulaspora delbrueckii with the sugar transporters of S. cerevisiae Hxt1 (NP 01 1962.1), Hxt3 (NP 01 0632.1), Hxt4 (NP 01 1960.2), Hxt5 (NP 01 1964.1), Hxt6 ( NP 01 0630.1), Hxt7 (NP 01 0629.3); K. lactis Kht1 (P18631.1) and Kht2 (P53387.1). Sc—Saccharomyces cerevisiae;Td—Torulaspora delbrueckii; Kl—Kluyveromyces lactis. The bootstrap consensus tree inferred from 500 replicates. adjacent in T. delbrueckii PYCC 5321 it is most probable that they IGT1 promoter has multiple biding sites for the are part of this cluster that includes two other potential trans- common hexose transporter transcription factors, Rgt1, porter(s). Like the S. cerevisiae HXT3–HXT6–HXT7 cluster, the T. Mig1 and Gcr1 delbrueckii cluster is adjacent to the SVF1 gene suggestive of a shared ancestral origin of the HXT and T. delbrueckii genes (www. Analysis of the IGT1 upstream sequence (1025 bp upstream ygob.ucd.ie)(Fig.3A). Consistent with the fact that T. delbrueckii from the ATG), carried out through the scanner tool of the diverged from S. cerevisiae before the WGD event (Wolfe and YeTFaSCo database, showed the presence of several potential Shields 1997), T. delbrueckii lacks the second HXT cluster (HXT5– Rgt1, Mig1, Gcr1-binding sites (Fig. S1, Supporting Information). HXT1–HST4) present in S. cerevisiae. The yeast Kluyveromyces lactis The transcriptional factors Mig1 and Rgt1 have been reported also has known glucose transporters in this locus, KHT1 (RAG1) as required for the transcriptional regulation of hexose trans- and KHT2 so we performed a phylogenetic analysis to deter- porters in S. cerevisiae. Mig1 mediates the repression of HXT2 and mine the evolutionary relationship of these transporters in dif- HXT4 expression at high concentrations of glucose, while Rgt1 ferent species (Fig. 3B). In the tree, Igt1 and Lgt1 cluster together acts as a bifunctional regulator of HXT1–HXT4 (Ozcan and John- in a larger clade that includes Hxt6/Hxt7 and the WGD ortho- ston 1996; Kim, Polish and Johnston 2003) but exclusively as a logue Hxt4. This pattern suggests that IGT1 and LGT1 arose repressor of expression of T. delbrueckii LGT1 gene (Alves-Araujo´ from a duplication in T. delbrueckii of the ancestral HXT gene in et al. 2005). Sequence analysis of the region upstream of IGT1 also this locus. The genes encoding the other putative transporters, showed the presence of a potential TATA box at position − 132, TDEL 0E02280 and TDEL 0E02290 also are likely to have arisen from the ATG codon, elements are often found in yeast promot- from a gene duplication event, and in this case cluster with Hxt3 ers, playing a critical role in transcription (Struhl 1982). Using and related transporters (Fig. 3B). Together the synteny (Fig. 3A) MEME (Motif-based sequence analysis tools) (Bailey and Elkan and phylogeny (Fig. 3B) are consistent with the idea that dupli- 1994) to compare IGT1 with LGT1 and HXT2 promoter regions, cation of the ancestor of HXT3 gave rise to TDEL 0E02280 and three common motifs with an E-value < 1werefound(TableS1, TDEL 0E02290 and that IGT1 and LGT1 arose from duplications of Supporting Information). We verified through gene ontology for the HXT7 ancestor. The orthologous genes in K. lactis are KHT1 motifs (GOMO) (Boden and Bailey 2008) that all three motifs are and KHT2, respectively. related to transmembrane transport. 6 FEMS Yeast Research, 2020, Vol. 20, No. 1 Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020

Figure 4. Glucose transport mediated by Igt1 in cells grown at different glucose concentrations. (A), Michaelis–Menten plot of glucose initial uptake rates in cells of S. cerevisiae hxt null strain transformed with the IGT1 gene. Cells were grown in SD 0.1% ()2%(•) and 4% () glucose without leucine, and harvested during exponential growth (OD640 = 0.5–0.6). (B), Eadie–Hofstee plots of glucose initial uptake rates for cells grown in SD 0.1% glucose (). (C), Kinetic parameters of glucose initial uptake in the cells described above. Where the data fitted more accurately to two transport components, the kinetic parameters of both components are given.

IGT1 encodes a dual-affinity glucose transporter

To carry out functional analysis, we sub-cloned the whole IGT1 ORF, 590 bp of the promoter and 497 bp of the terminator into YEplac181, creating the plasmid YEpIgt1. This was then transformed into S. cerevisiae EBY.VW4000 (hxt-null strain) to measure 14C-glucose uptake. As a control, S. cerevisiae EBY.VW4000 carry- ing the empty YEplac181vector was grown in SD-maltose and transferred to SD-glucose for 4 h. Under these conditions the control strain did not exhibit any measurable glucose uptake. Introduction of IGT1 into the hxt null strain was sufficient to allow it to grow on glucose, fructose and mannose (2% (w/v). Growth on glucose was also observed at 0.1% (w/v) and 4% (w/v) glucose, suggesting that Igt1 is expressed and enables transport at both high and low-glucose concentrations. Cells of S. cere- Figure 5. Eadie–Hofstee plots of glucose initial uptake rates in the presence of visiae hxt null strain transformed with IGT1, grown in SD-glucose other sugars in cells of S. cerevisiae hxt null strain transformed with the IGT1

2% (w/v), exhibited carrier-mediated glucose transport that best- gene. Cells were grown in SD-glucose (without leucine) (OD640 = 0.5–0.6). Zero fitted monophasic Michaelis–Menten kinetics (Fig. 4A), with an trans-influx of 14[U- C]glucose was measured in the absence (), and presence of 100 mM mannose (•) or 100 mM fructose (). apparent Km value of 11.5 ± 1.5 mM (intermediate-affinity), and aVmax of 1.81 ± 0.08 nmol/s/mg dry wt (Fig. 4C). Surprisingly, when IGT1 expressing cells are grown in 0.1% (w/v) glucose, glucose transport best-fitted biphasic Michaelis–Menten kinet- growth assays, glucose transport by Igt1 was competitively inhibited by fructose (Fig. 50), indicating that this sugar shared ics, with an intermediate (Km = 6.5 ± 2.0 mM) and a high- the Igt1 transporter. In the presence of 100 mM fructose, the affinity (Km = 0.10 ± 0.01 mM) component (Fig. 4B and C). Cells ± grown in glucose 4% (w/v), again exhibited kinetics of glucose apparent Km for glucose increased to 44.2 9.6 mM, with the transport best fitting one affinity component with an appar- Vmax remaining constant. The presence of 100 mM mannose only slightly inhibited glucose transport (Fig. 5), with the Km ent Km = 12.2 ± 2.6 mM, and a Vmax of 0.90 ± 0.09 nmol/s/mg dry wt (Fig. 4A and C). Although not frequent among hexose and Vmax values remaining almost unaltered (data not shown), transporters, this behavior is similar to what was described for suggesting that either it does not interact with the transporter S. cerevisiae Hxt2, which can display affinities that range from or that it is a very low affinity interaction. The fact that this 1.5 mM to 6.0 mM, depending on the glucose concentration strain is able to grow on mannose, seems to favor the last (Reifenberger, Boles and Ciriacy 1997). In agreement with the hypothesis. Pacheco et al. 7 Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020

Figure 6. Igt1 transmembrane domain and 3D structure predictions. (A), Amino acid sequence alignment between the T. delbrueckii proteins Lgt1 and Igt1. Residues that are similar in both proteins are marked with a point and the mismatched residues are specified. The numbers mark the amino acid positions. Identities = 481/557 (86%). The 12 transmembrane domains of Igt1 are highlighted by different colors. (B), Cartoon representation of Igt1p structure modelled to the XylE (I-TASSER), view parallel to the membrane. (C), The transmembrane domains of Igt1 are indicated from I to XII and from N to C terminus. Figures were prepared with program Pymol v.2.3 (DeLano 2002). (D), Igt1-binding site. Amino acid residues within 4 A˚ from the ligand (glucose in white) and the conserved motif GGFXXFGWD are reported. (E), Lgt1-binding site. C446 and W471 residues are reported in red, N366 is reported in yellow.

Prediction of Igt1 Structure template for the structure prediction and the study of the binding site. IGT1 encodes a predicted protein of 569 amino acids. Compari- Based on the evidence of the alignment and the 3D-structure son of the amino acid sequences of Igt1 and Lgt1 showed that model prediction, we could confirm that the protein consists of differences between the two proteins are mainly in two regions 12 transmembrane domains, reported in Fig. 6B and C. Beside the at their N-terminal end, between positions 4–48 and 140–166 transmembrane domains, the modelled structure shows a par- (Fig. 6A). ticularly extensive loop, which includes a small α-helical region, The membrane topology of Igt1 was deduced using sev- at the cytosolic side of the membrane between TMDs 6 and 7 eral programs. Depending on the program used, it was pre- (indicated with a green arrow in Fig. 6B). As recently reported by dicted to have either 10 (by TMHMM 2.0 SOSUI (Hirokawa, Singh et al. (Singh et al. 2008) and commented by Diallinas (Dialli- Boon-Chieng and Mitaku 1998) and TMPRED (Stoffel 1993)) nas 2008), this small α-helix may correspond to gating elements, or 12 (by PHOBIUS (Kall, Krogh and Sonnhammer 2004)and amino acids or parts of helices, located over and below the sub- POLYVIEW (Porollo, Adamczak and Meller 2004) membrane- strate binding site, acting as primary substrate binding sites. The spanning domains (Table S2, Supporting Information). When 3D prediction also shows the cytosolic localization of the N- and compared with transporter proteins that are predicted to have 12 C-terminals, as usual in many transporters. transmembrane domains, the 3th and 7th domains are missing Igt1 protein shows a sequence motif in the first transmem- in the 10 TMD predictions of Igt1. Indeed, the weaker hydropho- brane domain TMD1, which is conserved in several sugar trans- bicity of TMD3 and TMD7 makes these regions borderline and porters and may be involved with the specificity of the pro- not picked by some software. These transmembrane domain are tein (Knoshaug et al. 2015). Alignment of several MFS proteins reported to be involved in substrate binding or substrate translo- shows clear differences in this motif group, based on sugar cation across the membrane (Kruckeberg 1996; Ozcan and John- transport preferences of the transporters. Igt1 has the specific ston 1999), which could mean a less extensive contact with the conserved motif GGFXXFGWD (Fig. 6D in blue) of the transporter lipid bilayer. group which strongly favors glucose. Likewise, Igt1 shows the To get further insights into Igt1 structure, we built a 3D model YYX(T/P) motif in TMD7, which has no direct contact with the prediction of Igt1 using the I-TASSER platform (see methods). ligand, but it is involved in the change of the pore structure and Considering all the parameters provided by the server, the pre- it has also been shown to influence sugar specificity, favoring dicted model for Igt1 was considered to be adequate with a C- glucose (Wang, Yu and Zhao 2016). This is in agreement with the − ± score of 1.08, estimated TM-score 0.58 0.14 and a root mean competition studies were glucose was clearly preferred to fruc- ± ˚ square deviation (RMSD) of 10.1 4.6 A. The E. coli MFS pro- tose or mannose (Fig. 4). These two motifs are also conserved in ton:xylose symporter XylE is one of the top 10 proteins from the Lgt1 (Fig. 6E). PDB with close structural similarity to Igt1 and the X-ray struc- The amino acid residues within 4Ainvolvedintheinterac-˚ ture of this protein with glucose bound has been solved. This tion with the ligand (glucose in white) and the conserved motif structure, 4GBZ (10.2210/pdb4GBZ/pdb) was therefore used as GGFXXFGWD are reported in Fig. 6D. In the 3D model prediction 8 FEMS Yeast Research, 2020, Vol. 20, No. 1

of Igt1, TMD7 is in a central position and has a closer interaction The close sequence and 3D structure similarity but diver- with the substrate through the hydrophilic amino acids 331-QQ- gent kinetics between Lgt1 and Igt1, make them a very nice new 332 and 335-DN-336 (Fig. 6D in yellow). These residues are highly system to study structure/function relationships in eukaryotic conserved in the binding site of sugar transporters. When com- sugar transporters that may modulate their affinity. paring with the 3D model prediction of Lgt1, we found that the The results here presented may open the way to the improve- residues in the binding pocket are conserved, the only difference ment of sugar transport in T. delbrueckii and potentiate the being in the size of the pocket, which is tighter in Lgt1 due to the exploitation this emergent yeast species in biotechnology indus- presence of three more residues N366, C446 and W471 closer to try. Further studies are now needed to understand the physio- Downloaded from https://academic.oup.com/femsyr/article-abstract/20/1/foaa004/5715911 by University College Cork user on 10 February 2020 the ligand (Fig. 6E). This constrain in the binding site may hence logical role of Lgt1 and Igt1, and to characterize the other two underlie the lower affinity of Lgt1. potential hexose transporters.

DISCUSSION SUPPLEMENTARY DATA

In this study, we characterized a new glucose transporter in Supplementary data are available at FEMSYR online. T. delbrueckii, named Igt1 for intermediate-affinity glucose transporter, with a deduced structure sharing several features with members of the MFS (Marger and Saier 1993). Uptake kinetics ACKNOWLEDGMENTS obtained for this transporter, expressed in the hxt-null S. cerevisiae strain EBYVW.4000, demonstrate that this protein can We thank E. Boles (Goethe University Frankfurt, Germany) for function independently as a hexose transporter, encoding an the generous gift of strain S. cerevisiae EBY.VW4000 and George intermediate or a dual-affinity glucose transporter, depending Diallinas (University of Athens, Greece) for helpful discussions on the glucose concentration in the growth medium. A dual- on Igt1 structure. affinity transporter had been reported for S. cerevisiae Hxt2 and for Arabidopsis nitrate transporter Chl1. In the case of Chl1, FUNDING switching between the two modes of action is regulated by phos- phorylation at threonine residue 101; when phosphorylated, AP was supported by PhD fellowship [BD/13 282/2003] from Chl1 functions as a high-affinity nitrate transporter, whereas, Fundac¸ao˜ para a Cienciaˆ e para a Tecnologia (FCT), Por- upon dephosphorylated, it functions as a low-affinity nitrate tugal. LD is supported by the YEASTDOC Marie Curie ITN transporter (Liu and Tsay 2003). Igt1 has several potential phos- project which received funding from the European Union’s phorylation sites, which are not present in Lgt1 (predicted using Horizon 2020 Research and Innovation Programme under the NetPhosYeast 1.0 Server–Technical University of Denmark). the Marie Skłodowska-Curie grant agreement no 764 927. One of these residues could have an equivalent role to the ser- This work was supported by the strategic programme ine 101 residue of Chl1, being responsible for the alteration in UID/BIA/0 4050/2013 (POCI-01-0145-FEDER-0 07569) and the dual kinetic behavior of the Igt1 transporter, not observed project PTDC/BIA-MIC/32 059/2017 funded by national funds for Lgt1. The potential that there is a regulatory switch and inter- through the FCT,I.P. and by the ERDF through the COMPETE2020– play between two distinctive affinity modes makes further study Programa Operacional Competitividade e Internacionalizac¸ao˜ of this dual affinity transporter very interesting. (POCI) and Sistema de Apoio aInvestigac` ¸ao˜ Cient´ıfica e In addition to Igt1, it is likely that T. delbrueckii has still other Tecnologica´ (SAICT). high affinity transporters. This is supported by the kinetics of Conflict of interest. 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