The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene

Carmen-Lisset Flores{dagger}, Oscar H. Martínez-Costa{dagger}, Valentina Sánchez, Carlos Gancedo and Juan J. Aragón

Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain

Correspondence
Juan J. Aragón
juanjose.aragon{at}uam.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The phosphofructokinase from the non-conventional yeast Yarrowia lipolytica (YlPfk) was purified to homogeneity, and its encoding gene isolated. YlPfk is an octamer of 869 kDa composed of a single type of subunit, and shows atypical kinetic characteristics. It did not exhibit cooperative kinetics for fructose 6-phosphate (Hill coefficient, h 1·1; S0·5 52 µM), it was inhibited moderately by MgATP (Ki 3·5 mM), and it was strongly inhibited by phosphoenolpyruvate (Ki 61 µM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The gene YlPFK1 has no introns, and encodes a putative protein of 953 aa, with a molecular mass consistent with the subunit size found after purification. Disruption of the gene abolished growth in glucose and Pfk activity, while reintroduction of the gene restored both properties. This indicates that Y. lipolytica has only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of the Ylpfk1 disruptant in permissive carbon sources. The unusual kinetic characteristics of YlPfk, and the intracellular concentrations of glycolytic intermediates during growth in glucose, suggest that YlPfk may play an important role in the regulation of glucose metabolism in Y. lipolytica, different from the role played by the enzyme in Saccharomyces cerevisiae.


Abbreviations: Fru-6-P, fructose 6-phosphate; Fru-1,6-P2, fructose 1,6-bisphosphate; Fru-2,6-P2, fructose 2,6-bisphosphate; h, Hill coefficient; Pfk, phosphofructokinase; YlPfk, Pfk from Yarrowia lipolytica; ScPfk, Pfk from Saccharomyces cerevisiae

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY142710.

{dagger}These authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yarrowia lipolytica is an obligate aerobic non-conventional yeast that has attracted much attention because of some important characteristics. Among them are its unusual ability to grow on hydrocarbons, its ability to excrete important organic acids, its potential as a host for the expression of heterologous proteins (for reviews see Barth & Gaillardin, 1996), and its ability to shift between a yeast and a hyphal form (Domínguez et al., 2000). In spite of its basic and industrial interest, biochemical studies on its central metabolic pathways are scarce (Flores et al., 2000). Regarding the regulatory steps of glycolysis, a pathway nearly ubiquitous in all life forms, it is known only that the hexokinase activity of Y. lipolytica is low, about 1/10 of that of Saccharomyces cerevisiae (Petit & Gancedo, 1999), and that it is strongly inhibited by trehalose 6-phosphate (Petit & Gancedo, 1999), and that pyruvate kinase exhibits kinetic and regulatory properties significantly different from those of the enzyme from S. cerevisiae (Hirai et al., 1975). Nothing is known about the characteristics of Pfk (phosphofructokinase; EC 2.7.1.11), the enzyme that catalyses the physiologically irreversible phosphorylation of Fru-6-P (fructose 6-phosphate) to Fru-1,6-P2 (fructose 1,6-bisphosphate). The activity of this enzyme in different organisms is affected by a large variety of effectors, thus making Pfk an example of a ‘multimodulated enzyme’ (for reviews, see Uyeda, 1979; Sols, 1981; Kemp & Foe, 1983). The diversity of effectors that influence the activity of Pfk in different organisms has made it a qualified candidate to play a critical role in the regulation of glycolysis. In fact, in the yeast S. cerevisiae, it was thought to be the ‘bottleneck’ of this pathway; however, this view has been shaken by the fact that overproduction of the two subunits of S. cerevisiae Pfk did not alter the fermentation rate of this yeast (Davies & Brindle, 1992). Although the regulatory hierarchy of Pfk as controller of the yeast glycolytic pathway is still unsettled, its regulability, i.e. its capacity to be affected by different effectors (Hofmeyr & Cornish-Bowden, 1991), in a variety of organisms is undisputed, and the importance of its regulatory properties is shown by other results (Tornheim, 1988; Ogushi et al., 1990; González-Mateos et al., 1993). In addition to its role as a glycolytic enzyme, other functions have recently been described for Pfk in some organisms, such as its ability to modulate glucose-induced microautophagy in Pichia pastoris (Yuan et al., 1997), and to affect tubulin dynamics in the slime mould Dictyostelium discoideum (Orosz et al., 1999).

The metabolic peculiarities of Y. lipolytica, a yeast which diverged early from other yeasts (Souciet et al., 2000), make it interesting to study the kinetic and regulatory characteristics of the Pfk from this organism, as well as the physiological effects of the disruption of its encoding gene(s). In the work described here, we purified to homogeneity Pfk from Y. lipolytica, and characterized its physical and regulatory properties, which are discussed with regard to the particular metabolic regulation of this organism. We also demonstrate that there is only one gene encoding this protein, and show that it is essential for growth in glucose.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
All chemical reagents and enzymes used in genetic assays and protein purification were obtained from Roche Molecular Biochemicals, Biotools, Amersham Biosciences or Sigma. The auxiliary enzymes and biochemicals for the Pfk assay were from Sigma.

Organisms and growth conditions.
The following strains of Y. lipolytica were used in this study, as indicated where appropriate: CJM244 (MATA lys11-23 ura3-302 leu2-270) provided as INAG 35667 by A. Domínguez (Instituto de Microbiología Bioquíminca, Salamanca, Spain), and P01a (MATA leu2-270 ura3-302). The following S. cerevisiae strains were used: VW1a (MAT{alpha} ura3-52 leu2-3,112 his3{Delta}1 trp1-289 MAL2-8c SUC2 GAL) and the pfk1 pfk2 mutant HD152-1D (MAT{alpha} pkf1 : : HIS3 pfk2 : : HIS3 ura3-52, his3{Delta}1 leu2-3,112 trp1-289 MAL2-8c SUC2 GAL) provided by J. Heinisch (Universität Osnabrück, Germany). Yeasts were grown at 30 °C in 1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (w/v) glucose (YPD), or in minimal medium YNB (Difco), with the carbon sources indicated in each case at 2 % (w/v). Liquid cultures were shaken at 200 r.p.m. in a New Brunswick orbital shaker. Solid media were identical to liquid, but with 2 % (w/v) agar added. Auxotrophic requirements were added when necessary at 20 µg ml–1. Transformation of Y. lipolytica was done as described by Barth & Gaillardin (1996), and that of S. cerevisiae as described by Ito et al. (1983). The Escherichia coli strain DH5{alpha} [supE44 {Delta}lacU169 ({pi}80 lacZ{Delta}M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1], used for general cloning procedures and amplifications of DNA, was grown at 37 °C in either liquid or solid Luria–Bertani medium (Sambrook et al., 1989), with 50 µg ampicillin ml–1 when necessary.

Purification of Pfk.
All purification steps were performed at 4 °C. Cells from a 2 l culture of Y. lipolytica CJM244 grown in YPD to stationary phase were harvested by sedimentation, washed twice in cold water, and then washed in buffer A (50 mM HEPES, 100 mM KCl, 2 mM EDTA, 1 mM DTT, 2 mM NaF, 0·5 mM PMSF, 2·5 µg leupeptin ml–1, pH 6·8). Approximately 15 g (wet weight) cells were resuspended in 3 vols buffer A, and disrupted with 5 vols glass beads, as described previously (Aragón et al., 1986). Cell debris was removed by centrifugation at 10 000 g for 10 min, followed by centrifugation of the resulting supernatant at 31 000 g for 30 min. Protamine sulfate dissolved in buffer A was added to the supernatant to a final concentration of 0·2 % (w/v). After 30 min stirring, and centrifugation as before, the supernatant fluid, designated the extract, was precipitated with 5 % (w/v) solid polyethylene glycol (Mr 8000). After 30 min stirring, and centrifugation as described above, the resulting pellet was dissolved in buffer B [50 mM HEPES, 5 mM MgCl2, 2 mM EDTA, 1 mM DTT, 0·5 mM PMSF, 2·5 µg leupeptin ml–1, 20 % (v/v) glycerol, pH 6·8], and separated by chromatography on a column (2·2x8·0 cm) of DEAE-cellulose (DE52) equilibrated with buffer B. The column was washed with 120 ml buffer B, and eluted with a 200 ml linear gradient of 0–500 mM KCl in buffer B. Fractions of 2 ml were collected, and analysed for protein and Pfk activity. YlPfk eluted at approximately 180 mM KCl, and the more active fractions were pooled and applied to a column (1·9x7·0 cm) of Blue Sepharose CL-6B equilibrated with buffer B. The column was washed with 60 ml buffer B, 50 ml buffer B containing 3 mM Fru-6-P and 1·5 mM MgATP, and eluted in 2 ml fractions with a 100 ml linear gradient of 0–1·5 M KCl in buffer B. Pfk was eluted at approximately 0·75 M KCl, and fractions having the highest activity were pooled and employed in subsequent studies. The final purified preparation had a specific activity of 10·5 U (mg protein)–1. This preparation was subjected to SDS-PAGE analysis on 10 % gels, stained with Coomassie blue as previously described (Martínez-Costa et al., 1994), and judged to be homogeneous by this criterion (see Results). For comparative kinetic studies between YlPfk and ScPfk, the cell extracts were dialysed against 10 mM HEPES, 0·5 mM PMSF, 2·5 µg leupeptin ml–1 and 20 % (v/v) glycerol, pH 7·0.

Enzyme activity assay.
Total Pfk activity was measured in an assay mixture containing 50 mM HEPES, 100 mM KCl, 5 mM MgCl2, pH 7·0, 2 mM Pi, 0·4 mM {mic1511465E001}, 40 µM cAMP, 0·15 mM NADH, 1 mM MgATP, 1·2 U aldolase, 10 U triosephosphate isomerase, 1 U glycerol-3-phosphate dehydrogenase, 5–10 µl enzyme preparation and 2 mM Fru-6-P, in a final volume of 1 ml. Assays for kinetic studies were carried out under the same conditions, omitting Pi, {mic1511465E002} and cAMP unless otherwise stated, with 5–10 µl purified enzyme, and the indicated concentrations of MgATP, the corresponding effector and Fru-6-P. When the effect of citrate was studied, the concentration of MgCl2 was 20 mM. In all cases, the reaction mixture without Fru-6-P was preincubated for 5 min. The reaction was started by the addition of Fru-6-P, and followed by measuring the rate of the absorbance change at 340 nm at 25 °C. When Pfk activity was assayed during purification, glucose-6-P was added to Fru-6-P in a 3 : 1 molar proportion. Auxiliary enzymes were desalted as described previously (González-Mateos et al., 1993; Martínez-Costa et al., 1994). One unit (U) of activity is defined as the amount of enzyme that catalyses the conversion of 1 µmol substrate min–1 under the conditions described above.

Size-exclusion FPLC.
Isocratic size-exclusion chromatography was performed at 20 °C on a Superdex 200 HR 10/30 column (Amersham Biosciences). The column was equilibrated with 50 mM sodium phosphate buffer, pH 7·0, containing 150 mM NaCl. Samples of 0·2 ml were applied to the column, and eluted at a flow rate of 0·5 ml min–1 with the same buffer. Fractions of 0·1 ml were collected, and tested for Pfk activity. When indicated, the elution buffer contained 10 mM Fru-6-P. For calibration, standard proteins ranging from 13·7 to 880 kDa were used.

Isolation of the YlPFK1 gene.
Degenerate oligonucleotides were designed against two regions of Pfk in which conserved amino acid sequences were found in diverse organisms. The design took into account the high G+C content of the Y. lipolytica genome (http://www.kazusa.or.jp/codon/). The oligonucleotides were GTCGGYTCYATYGACAACGACATG for the 5' region, and GTACTCGGTRCCGGGSACRTT for the 3' region (the standard abbreviations to represent ambiguity are used; Nomenclature Committee of the International Union of Biochemistry, 1985), corresponding to VGSIDNDM and NVPGTEY, respectively. A PCR reaction was performed using these oligonucleotides, and Y. lipolytica genomic DNA as a template. The conditions were: 30 cycles of 1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C. A DNA fragment of about 1 kb was isolated, cloned into pGEMT-easy (Promega), and sequenced. The translated sequence of this fragment was highly similar to that of other Pfks. To obtain the rest of the coding region, 5' and 3' RACE reactions were performed using the RLM-RACE kit (Ambion; catalogue no. 1700) and the following oligonucleotides: for the 5' RACE, inner primer TCGACGACAAAGGCTCGCGAGTGC, and outer primer CCCACTGAGTAGCATCGGGAGCAG, and for the 3' RACE, CAACGGTTTGACGGTCTAATCGTC. For the 5' RACE reaction, the conditions were as follows: three cycles of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C; 30 cycles of 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C; followed by a cycle of 1 min at 94 °C, 1 min at 60 °C, and 5 min at 72 °C. For the 3' RACE, the following conditions were used: 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1·2 min at 72 °C; followed by one cycle of 1 min at 94 °C, 1 min at 50 °C, and 5 min at 72 °C. The products of the reaction were cloned into pGEMT-easy, and sequenced. All the products were sequenced at least twice in each direction. The sequence corresponding to the coding region of the YlPFK1 gene encoding the Y. lipolytica Pfk has been deposited in the GenBank database under the accession number AY142710.

To obtain a DNA fragment comprising the whole coding region of YlPFK1, the following oligonucleotides were used: for the 5' region, CGCGCTTCAAAACACCGACAAT, and for the 3' region, AATTTCCAACACCAAACCCCCTA. A PCR reaction was done using genomic Y. lipolytica DNA and the following conditions: three cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C; 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 3 min at 72 °C; followed by 1 min at 94 °C, 1 min at 48 °C, and 5 min at 72 °C. The product was cloned into pGEMT-easy to yield plasmid pCL79, and sequenced.

Expression of YlPFK1 in Y. lipolytica and S. cerevisiae.
YlPFK1 was expressed in Y. lipolytica using the plasmid pCL49, which carries the YlTEF1 promoter and the YlXPR2 terminator (Flores & Gancedo, 2005). The 3 kb YlPFK1 ORF was excised from pCL79 with NotI, and cloned in the same site in pCL49. To express YlPFK1 in S. cerevisiae, a monocopy plasmid, pCL80, and a multicopy plasmid, pCL81, were constructed. Both plasmids carried the coding region of the YlPFK1 gene under the control of the S. cerevisiae ADH1 promoter, and they were constructed as follows. Plasmid pCL80 was obtained by inserting in the BamHI site of pRS316 (Sikorski & Hieter, 1989) a cassette carrying the promoter and terminator of ADH1 excised from plasmid pAAH5 (Ammerer, 1983), and then introducing in the blunt-ended HindIII site the PCR fragment carrying YlPFK1 (obtained using Pwo DNA Polymerase; Roche). Plasmid pCL81 was created by cutting plasmid pDB20 (Becker et al., 1991) with NotI, and inserting the 3 kb NotI–NotI fragment from pCL79 carrying YlPFK1.

Disruption of the chromosomal YlPFK1 copy.
To disrupt the YlPFK1 gene, plasmid pCL79 was cut with NotI, and the fragment containing the YlPFK1 gene was inserted in the NotI site of a pKS plasmid (Stratagene) lacking the piece between XbaI and KpnI in the polylinker. The resulting construction was digested with NcoI, and the internal 1·66 kb NcoI–NcoI fragment from YlPFK1 was replaced by a DNA fragment of 2·1 kb containing the YlLEU2 gene obtained by NcoI digestion of plasmid pINA62 (Gaillardin & Ribet, 1987) to give plasmid pCL83. The 3·4 kb NotI–NotI fragment from pCL83 was used to transform Y. lipolytica. Transformants were selected by growth in the absence of leucine on minimal medium agar containing 3 % glycerol and 2 % ethanol, and replica plated to minimal medium glucose agar. Those transformants unable to grow on glucose were selected for further study.

Nucleic acid manipulations.
Recombinant DNA techniques were done according to established protocols. DNA sequencing was performed by the dideoxy chain-termination method. Sampling of the yeast to extract RNA was done by rapid filtration and flash freezing in liquid nitrogen, as described by Belinchón et al. (2004). Total RNA for RACE reactions was extracted from Y. lipolytica using the TRIzol LS reagent (Invitrogen). Southern analysis was performed using established protocols. Probes were labelled with 32P using the Rediprime II Random Prime labelling system. Genomic DNA was obtained as described by Hoffman & Winston (1987).

Other methods.
Protein concentration was determined by Bradford's dye-binding method (Bradford, 1976), using bovine {gamma}-globulin as the standard. Yeasts were sampled during the exponential phase of growth, and metabolite extraction was done as described previously (Gamo et al., 1993). Metabolites were determined by standard enzymic techniques (Bergmeyer et al., 1987). Multialignments of Pfk sequences were conducted by means of the program CLUSTAL W (Higgins et al., 1994).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purification and molecular properties of Y. lipolytica Pfk
Cell-free extracts of glucose-grown Y. lipolytica CJM 244 had a Pfk activity of 63±8 mU (mg protein)–1, corresponding to a total activity of 6±1 U (g wet weight)–1. This value was about threefold lower than that shown by extracts of glucose-grown S. cerevisiae done in parallel [18·5±1·8 U (g wet weight)–1]. YlPfk was purified by the procedure summarized in Table 1. As shown, two effective steps, namely ion-exchange chromatography on DE52, and affinity chromatography on Blue Sepharose, led to a pure enzyme preparation with a specific activity of 10·5 U (mg protein)–1. The final preparation showed only one band in SDS-PAGE (Fig. 1a), indicating that it was homogeneous, and that YlPfk is likely to be composed of a single type of subunit of molecular mass 109±2 kDa.


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Table 1. Purification of Y. lipolytica Pfk

 


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Fig. 1. (a) SDS-PAGE and (b) size-exclusion FPLC of purified Y. lipolytica Pfk. (a) A 1 µg quantity of purified enzyme was loaded on the gel. The positions and molecular masses of marker proteins are indicated on the right. (b) Elution was carried out in the presence of 10 mM Fru-6-P. Enzyme activity was measured at 2 mM Fru-6-P, 1 mM MgATP. Arrows indicate the theoretical elution volumes of YlPfk forms corresponding to octamer, tetramer, dimer and monomer, and their calculated Mr. Inset, native molecular mass determination of YlPfk. Molecular size standards: a, the 48-subunit form of ferritin, 880 kDa; b, thyroglobulin, 669 kDa; c, the 24-subunit form of ferritin, 440 kDa; d, catalase, 232 kDa; e, aldolase, 158 kDa; f, albumin, 67 kDa; g, ovoalbumin, 43 kDa; h, chymotrypsinogen A, 25 kDa; i, ribonuclease A, 13·7 kDa. Addition of Fru-6-P to the chromatography buffer was necessary to elute the enzyme in an active form.

 
When the purified enzyme was subjected to FPLC size-exclusion chromatography on Superdex 200HS 10/30, Pfk activity eluted as a single peak (Fig. 1b) with a molecular mass of 869±26 kDa, suggesting that the native enzyme is an octamer of identical subunits.

Kinetic and regulatory properties of Y. lipolytica Pfk
We studied different kinetic properties of YlPfk using the purified enzyme, obtained as described above, and dialysed cell extracts from Y. lipolytica and S. cerevisiae to compare some parameters of the enzymes from both origins. As shown in Fig. 2(a), YlPfk exhibited a high affinity for Fru-6-P (S0·5 52 µM), and no evidence of cooperativity for this substrate was found [h (Hill coefficient) 1·1]. This contrasts with the sigmoidal curve and S0·5 values within the mM range shown by Pfks of most organisms; compare, for example, the h and S0·5 values of ScPfk with those of YlPfk (2·8 and 1·48 mM versus 1·04 and 90 µM, respectively). Also, YlPfk was only slightly inhibited by MgATP (Fig. 2b), showing a Ki value of 3·5 mM, about 10-fold higher than that of the S. cerevisiae Pfk (Fig. 2b, inset). An increase in the MgATP concentration from 1 to 5 mM brought about an increase of the S0·5 value of YlPfk for Fru-6-P to 0·51 mM, as expected, but the saturation curve for this substrate remained hyperbolic (h 1·1) (results not shown). The calculated kcat value (152 s–1) is about one order of magnitude lower than those known for Pfks from other yeast species, such as S. cerevisiae (Hofmann & Kopperschläger, 1982), Schizosaccharomyces pombe (Reuter et al., 2000), P. pastoris (Kirchberger et al., 2002) and Kluyveromyces lactis (Bär et al., 1997).



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Fig. 2. Dependence of Y. lipolytica Pfk activity on the substrate concentration. Assays were carried out as described in Methods. (a) MgATP concentration was 1 mM. (b) Fru-6-P concentration was 25 µM. Insets, comparison of the kinetic behaviour of YlPfk ({bullet}) and ScPfk ({circ}) assayed in dialysed cell extracts. Conditions were as described above, except that the MgATP dependence of ScPfk was assayed at 1·5 mM Fru-6-P. Activity is expressed relative to the maximal activity obtained for each preparation.

 
The most remarkable difference in the behaviour of the YlPfk was its insensitivity to Fru-2,6-P2 (Fig. 3a), a strong activator of eukaryotic Pfks (Van Schaftingen, 1987). As can be seen in Fig. 3(a), the enzyme from S. cerevisiae was activated about 100-fold by saturating concentrations of Fru-2,6-P2 with a Kact (activation constant) value of 1·3 µM, while the enzyme from Y. lipolytica remained basically unaffected.



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Fig. 3. Effect of (a) fructose-2,6-P2 and (b) phosphoenolpyruvate on Y. lipolytica Pfk. Assays were carried out as described in Methods. Fructose-2,6-P2 activation was assayed at 50 µM Fru-6-P and 5 mM MgATP. Phosphoenolpyruvate (P-enolpyruvate) inhibition was assayed at 50 µM Fru-6-P and 3·5 mM MgATP. Insets, results with dialysed cell extracts from Y. lipolytica ({bullet}) and S. cerevisiae ({circ}). Conditions for YlPfk were as described above. The effect of Fru-2,6-P2 and phosphoenolpyruvate on ScPfk activity was studied in the presence of 5 mM and 0·5 mM MgATP, respectively, and at 1 mM Fru-6-P. Activity is expressed relative to the maximal activity obtained for each enzyme in the presence of Fru-2,6-P2 (a, inset) and in the absence of phoshoenolpyruvate (b, inset).

 
Phosphoenolpyruvate markedly inhibited YlPfk, with a Ki value of 61 µM, two orders of magnitude lower than the value found for ScPfk (Fig. 3b). With respect to other typical allosteric effectors of Pfk from other sources, the activity of YlPfk was affected as follows compared with the control [0·63±0·08 mU (mg protein)–1]: it was inhibited 80 % by 5 mM citrate [0·12±0·02 mU (mg protein)–1], activated three- to fourfold by 5 mM Pi [2·1±0·2 mU (mg protein)–1] and 5 mM {mic1511465E003} [2·4±0·1 mU (mg protein)–1], and not significantly affected by 5 mM ADP [0·43±0·04 mU (mg protein)–1] and 1 mM AMP [0·63±0·08 mU (mg protein)–1] (enzyme activities are reported as means±SEM of three to five determinations). The saturation curve for citrate was sigmoidal (h 2·1), with a Ki value of 3·3 mM; the curves for Pi and {mic1511465E004} were hyperbolic, with Kact values of 0·62 and 0·66 mM, respectively (results not shown).

Table 2 shows the concentrations of glycolytic metabolites related to Pfk function that we measured in Y. lipolytica, as compared with those determined in S. cerevisiae. The most striking result concerned the reaction product of Pfk, Fru-1,6-P2, whose intracellular concentration (80 µM) in cultures of Y. lipolytica actively growing in YPD was two orders of magnitude lower than that measured in S. cerevisiae under similar conditions (4·53 mM). The concentrations of other metabolites were within the same range in both species, although the ATP concentration was lower in Y. lipolytica.


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Table 2. Intracellular concentrations of selected metabolites in Y. lipolytica and S. cerevisiae grown in YPD

Yeasts were sampled as described in Methods. Concentrations (mM) of metabolites were obtained by multiplying the values in µmol (g wet weight)–1 by 2·5; this assumes that the intracellular water is 50 %, and that the wet weight/dry weight ratio is 5. Results represent the means (±SEM) of between three and five determinations of five independent cultures.

 
Isolation of the YlPFK1 gene, and comparison of the encoded protein with other Pfks
The gene encoding YlPfk was isolated as described in detail in Methods. Basically, degenerate oligonucleotides corresponding to two regions of conserved amino acid sequences in Pfks of diverse organisms were designed taking into account the characteristics of the codon usage of Y. lipolytica. A PCR reaction using genomic Y. lipolytica DNA as the template allowed the isolation of a piece of DNA of about 1 kb whose translated sequence was highly similar to that of other Pfks. Using 5' and 3' RLM-RACE reactions, the sequence corresponding to the entire coding region of the YlPFK1 gene was obtained. The structural gene does not exhibit introns, and its G+C content was close to 57·5 mol%, a result not surprising in view of the high G+C content of the Y. lipolytica genome (Dujon et al., 2004). The YlPFK1 gene encodes a putative protein of 953 aa, a value in agreement with the subunit size found in the purification. This protein presents a high sequence similarity with Pfks of other origins, showing around 50 % identity with Pfks from diverse organisms (e.g. 54 % with the protein of Giberella zeae, 53 % with Pfk2 from Candida albicans, 52 % with the enzyme from Sch. pombe, 48 and 49 % with the {alpha} and {beta} subunits, respectively, from S. cerevisiae, and as much as 43–44 % with Pfk isoenzymes from mammals). An alignment of the deduced amino acid sequence of YlPfk with those of Pfks from different organisms (Fig. 4) revealed that all functional domains containing amino acid residues assigned to binding sites for substrates and allosteric effectors (Poorman et al., 1984; Shirakihara & Evans, 1988; Martínez-Costa et al., 2004) are conserved in this Pfk.



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Fig. 4. Alignments between amino acid sequences of Pfks from different organisms. The sequences shown are from Y. lipolytica (Yl), Sch. pombe (Sp), S. cerevisiae (Sc), human muscle (Hm) and E. coli (Ec). Amino acids identical to YlPfk are shaded. Putative residues assigned to the binding of substrates and allosteric effectors (Poorman et al., 1984; Shirakihara & Evans, 1988; Martínez-Costa et al., 2004) are indicated by black triangles.

 
When expressed in S. cerevisiae under the control of the S. cerevisiae ADH1 promoter, either in mono- or multicopy plasmids, the YlPFK1 gene complemented the lack of growth in glucose of a S. cerevisiae pfk1 pfk2 mutant (results not shown).

Physiological effects of the disruption of the YlPFK1 gene
The chromosomal copy of YlPFK1 was disrupted by deletion of an internal 1·66 kb fragment and substitution by the YlLEU2 gene, as described in Methods. A Southern analysis showed the correctness of the disruption (Fig. 5a). Disruption of the YlPFK1 gene resulted in glucose-negative growth (Fig. 5b), and abolished detectable Pfk activity. The findings on protein structure, which indicated a protein composed of identical subunits, are in agreement with the findings obtained in the gene disruption. Reintroduction of the YlPFK1 gene restored growth on glucose (Fig. 5b), thus confirming that the inability to grow in glucose of the disruptant was caused by the lack of Pfk. Although glucose did not support growth of the Ylpfk1 disruptant, it did not interfere with growth on permissive carbon sources (Fig. 5b), which is in contrast with the situation observed in S. cerevisiae (C.-L. Flores & C. Gancedo, unpublished results).



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Fig. 5. Disruption of YlPFK1 gene in Y. lipolytica, and phenotypic analysis of the disruption. (a) Scheme of the YlPFK1 gene (white box), its disruption with the YlLEU2 gene (grey box), and Southern blot analysis of the disruption. Genomic DNA was digested with EcoRV, and probed with the indicated 439 bp NcoI–NotI fragment obtained from plasmid pCL79 (for details, see Methods). PO1a, wild-type; CLP5, strain with disrupted YlPFK1. (b) Phenotypic analysis of the disruptant. The disruptant strain CLP5 was transformed with plasmids carrying either the YlPFK1 gene with the correct orientation relative to the promoter, or with the reversed gene. Two independent transformants with a reintroduced YlPFK1 gene in the correct orientation, and one with the reversed orientation, are shown. The carbon sources of the corresponding agar plates are indicated; and the media were supplemented with leucine and uracil. The relevant characteristics of the strains are indicated in the diagram.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we isolated and characterized the Pfk from Y. lipolytica and its encoding gene. YlPfk was purified to homogeneity, and was found to consist of a single type of polypeptide chain of 109 kDa, the native enzyme being a homo-octamer of 869 kDa. The identical structure of the subunits was further supported by the lack of growth in glucose after disruption of the unique YlPFK1 gene, as well as by the functional complementation of a S. cerevisiae pfk1 pfk2 mutant. Additionally, the recent availability of the Y. lipolytica genome (http://cbi.labri.fr/Genolevures) shows that there is no other ORF with significant similarity to the one described in this paper. In contrast to Pfk from other organisms, which are tetrameric proteins, the enzymes from yeasts are octameric, except for the Rhodotorula glutinis Pfk, which was suggested to be a tetramer (Schröter & Kopperschläger, 1996). However, the subunit composition of yeast Pfks varies between species: while the enzymes from S. cerevisiae, K. lactis, P. pastoris and C. albicans are hetero-octameric proteins (Kopperschläger et al., 1977; Heinisch et al., 1993; Bär et al., 1997; Lorberg et al., 1999; Kirchberger et al., 2002), those of the fission yeasts Sch. pombe and Y. lipolytica are homo-octameric (Reuter et al., 2000; this work). Thus, the acquisition of an octameric structure does not necessarily result from the interaction between two different types of subunits. It is usually assumed that Pfk subunits from eukaryotes arose by duplication, fusion and mutation of an ancestral prokaryotic gene (Poorman et al., 1984), which, in the case of several yeast species, was followed by a second duplication event leading to two different genes (Heinisch et al., 1989). Phylogenetic analyses indicate that Y. lipolytica separated early from other yeast genera, after the branching of Sch. pombe (Kurtzman & Blanz, 1998; Souciet et al., 2000). Interestingly, the Pfks of Sch. pombe and Y. lipolytica are composed of a single type of subunit, thus making attractive the hypothesis that the appearance of a second gene encoding a different subunit occurred after the separation of these species. The possible physiological advantages, if any, of the gene duplication have not been addressed.

The kinetic study of the purified Pfk from Y. lipolytica, and the isolation of its encoding gene, have revealed important differences between this organism and other yeasts. Unlike the enzyme from S. cerevisiae and most other cells, YlPfk has a high affinity for Fru-6-P, and lacks co-operative interactions in the presence of this substrate, suggesting that the enzyme is primarily in the more active R conformation, as assumed by the model of Monod et al. (1965). Additionally, YlPfk shows little inhibition by MgATP, and is insensitive to the allosteric activators Fru-2,6-P2, AMP and ADP, whereas it is highly sensitive to inhibition by phosphoenolpyruvate. In bacteria, phosphoenolpyruvate is the main allosteric inhibitor of Pfk (Blangy et al., 1968), but in S. cerevisiae and animal cells, this compound is of little physiological relevance because of its intracellular concentration, which is usually several orders of magnitude lower than the Ki values of Pfk isozymes (inset of Fig. 3b, Table 2; Martínez-Costa et al., 2004). In contrast, our data suggest that phosphoenolpyruvate could play a physiological role in the regulation of YlPfk, as its intracellular concentration during growth in YPD (0·27 mM) is about fourfold higher than its Ki value for YlPfk (61 µM). Under these conditions, the inhibition by phosphoenolpyruvate would not allow the enzyme to work near Vmax, even if the concentration of Fru-6-P (0·19 mM), much higher than the S0·5 value (52 µM), would permit it. Therefore, phosphoenolpyruvate, probably in conjunction with the activators Pi and {mic1511465E005}, may contribute to maintaining YlPfk within a range of in vivo activity able to respond efficiently to variations of Fru-6-P concentration, even in the absence of co-operative kinetics. To our knowledge, no data are available on the effect of this metabolite on Pfks from other yeast species, except S. cerevisiae. It has been reported (Habison et al., 1983) that partially purified Pfk from Aspergillus niger is inhibited by phosphoenolpyruvate with a Ki value of 0·15 mM, and also by citrate (Ki 0·1–0·4 mM), the latter inhibition being released by Fru-2,6-P2 activation (Arts et al., 1987). Inhibition by citrate is a common feature of different eukaryotic Pfks; however, in Y. lipolytica, at least during growth in YPD, the role of this inhibitory effect is uncertain because of the high Ki value of 3·3 mM, and the undetectable intracellular concentration of citrate under these conditions.

The atypical kinetic properties of YlPfk, and the levels of key glycolytic intermediates determined in Y. lipolytica, point to a role of this enzyme in the regulation of glycolysis that is different from the role played in S. cerevisiae. The high concentration of Fru-1,6-P2 found in S. cerevisiae during growth in glucose, greatly exceeding the concentration of Fru-6-P (Table 2; Bañuelos et al., 1977; Breitenbach-Schmitt et al., 1984), suggests that in this yeast, a rate-limiting step of the glycolytic pathway is located after the reaction catalysed by Pfk. However, in Y. lipolytica, the situation appears to be different since the concentration of Fru-1,6-P2 in cultures growing in YPD (over 50-fold lower than that in S. cerevisiae) was about half that of Fru-6-P, thus suggesting that in this organism, Pfk may have an important role on the control of glucose utilization.

All the amino acid residues assigned to the binding of Fru-2,6-P2 and AMP/ADP are conserved in YlPfk (Fig. 4). Therefore, the lack of activation by these effectors could be related to other unidentified residues important for either the binding to their allosteric sites or the signal transduction route from these sites to the catalytic site. It is also possible that the insensitivity of YlPfk towards Fru-2,6-P2 may be due to an evolutionary adaptation to the lack of this compound; however, this seems unlikely. Although no data on Fru-2,6-P2 concentration in Y. lipolytica are available, two ORFs, YALI0E17633g and YALI0F27885g, have been found in the available Y. lipolytica genome (http://cbi.labri.fr/Genolevures) with similarity to S. cerevisiae 6-phosphofructose-2-kinase and fructose-2,6-bisphosphatase, respectively, the enzymes responsible for the metabolism of this compound.

Among eukaryotic Pfks with abnormal allosteric properties, the Sch. pombe enzyme was reported to have little sensitivity to ATP inhibition (Reuter et al., 2000), and that from K. lactis shows no Fru-6-P co-operativity (Bär et al., 1997); however, both enzymes are sensitive to usual Pfk effectors, including Fru-2,6-P2 and AMP. A nonallosteric Pfk has been described in the slime mould D. discoideum (Martínez-Costa et al., 1994).

S. cerevisiae mutants lacking Pfk activity do not grow in glucose because of limited functionality of the pentose phosphate pathway, and because of the fermentative sugar metabolism in this yeast. In contrast, in K. lactis, a yeast with a more oxidative metabolism than S. cerevisiae, pfk mutants grow in glucose at a rate similar to that of the wild-type (Heinisch et al., 1993). However, an additional block of respiration, or mutations that affect the function of the pentose phosphate pathway, precluded growth of K. lactis pfk mutants in glucose (Jacoby et al., 1993). In this context, the lack of growth in glucose of the Y. lipolytica pfk mutant is unexpected, since this yeast has an obligate oxidative metabolism, and even possesses an active complex I in the respiratory chain (Djafarzadeh et al., 2000). The most likely explanation of this growth phenotype would be a problem with the functionality of the pentose phosphate pathway, perhaps related to NADP+ regeneration. However, direct measurements of the glucose flux going through this pathway in Y. lipolytica are not yet available.

An interesting difference between the behaviour of Y. lipolytica pfk1 and S. cerevisiae pfk1 pfk2 mutants is their response to glucose. This sugar inhibits growth in media with gluconeogenic carbon sources not only of the S. cerevisiae pfk1 pfk2 mutant (C.-L. Flores & C. Gancedo, unpublished results), but also of other glycolytic mutants (Ciriacy & Breitenbach, 1979). In contrast, the growth of the Y. lipolytica pfk1 mutant under similar conditions remained unaffected by the sugar. This difference might be due to a variation in the magnitude of catabolite repression among the two yeasts. While glucose exerts a strong repression on pathways that allow the use of gluconeogenic carbon sources in S. cerevisiae (Gancedo, 1998; Carlson, 1999), this repression is less marked in Y. lipolytica (Barth & Scheuber, 1993; R. Jardón, C.-L. Flores & C. Gancedo, unpublished results). This fact, and the function of an active respiration, may allow the Y. lipolytica pfk1 mutant to withstand the presence of glucose.

The peculiar regulatory characteristics of the YlPfk, together with the differences in kinetic properties of pyruvate kinase (Hirai et al., 1975), and the low hexokinase activity found in this organism (Petit & Gancedo, 1999), indicate that the glycolytic flux in this organism is regulated in a different way from that described in the conventional yeast S. cerevisiae.


   ACKNOWLEDGEMENTS
 
We thank Drs J. M. Gancedo (this Institute) for critical reading and comments on the manuscript, and A. Domínguez (IMBQ, Salamanca, Spain) and C. Gaillardin (INRA, Grignon, France) for providing yeast strains or plasmids. This work was supported by grants from the Dirección General de Investigación del Ministerio de Ciencia y Tecnología BMC-1691-2001-CO2-01 (to C. G.), and PB98-0058 and BMC 2002-00769 (to J. J. A.), and Instituto de Salud Carlos III, Red de Centros C03/08 (to J. J. A.).


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Received 27 December 2004; revised 11 February 2005; accepted 15 February 2005.



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