A Novel Form of 6-Phosphofructokinase
2007; Elsevier BV; Volume: 282; Issue: 32 Linguagem: Inglês
10.1074/jbc.m611547200
ISSN1083-351X
AutoresKatrin Tanneberger, Juörgen Kirchberger, Joörg Baör, Wolfgang Schellenberger, Sven Rothemund, Manja Kamprad, Henning Otto, Torsten Schoöneberg, Anke Edelmann,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoClassically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of α subunits and α/β subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, α, β, and γ. The sequence of the γ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the γ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the γ subunit is not required for enzymatic activity, the γ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and γ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the γ subunit. Therefore, we suggest that the γ subunit contributes to adaptation of P. pastoris to energy resources. Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of α subunits and α/β subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, α, β, and γ. The sequence of the γ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the γ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the γ subunit is not required for enzymatic activity, the γ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and γ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the γ subunit. Therefore, we suggest that the γ subunit contributes to adaptation of P. pastoris to energy resources. The ATP-dependent 6-phosphofructokinase (EC 2.7.1.11, phosphofructokinase-1, ATP:d-fructose-6-phosphate 1-phosphotransferase (Pfk)) 2The abbreviations used are: Pfk, 6-phosphofructokinase; DIG, digoxigenin; FACS, fluorescence-activated cell sorting; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometer; PpPfk, 6-phosphofructokinase from P. pastoris; RACE, rapid amplification of cDNA ends; ScPfk, 6-phosphofructokinase from S. cerevisiae; Fru 6-P, fructose 6-phosphate; Fru 2,6-P2, fructose 2,6-bisphosphate; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting. catalyzes in many organisms the phosphorylation of fructose 6-phosphate (Fru 6-P) at position 1. The Pfk activity is generally being sensitive to a number of allosteric regulators, e.g. ATP, AMP, NH4+, and fructose 2,6-bisphosphate (Fru 2,6-P2). Therefore, this irreversible reaction is considered to be one of the rate-limiting steps of glycolysis (1Hofmann E. Rev. Physiol. Biochem. Pharmacol. 1976; 75: 1-68Crossref PubMed Google Scholar, 2Hofmann E. Kopperschlaöger G. Methods Enzymol. 1982; 90: 49-60Crossref PubMed Scopus (88) Google Scholar, 3Uyeda K. Adv. Enzymol. Relat. Areas Mol. Biol. 1979; 48: 193-244PubMed Google Scholar). Most eukaryotic Pfks are heteromeric enzymes consisting of subunits, which evolved from a single ancestor gene by gene duplication and mutational events (4Poorman R.A. Randolph A. Kemp R.G. Heinrikson R.L. Nature. 1984; 309: 467-469Crossref PubMed Scopus (184) Google Scholar, 5Heinisch J. Ritzel R.G. von Borstel R.C. Aguilera A. Rodicio R. Zimmermann F.K. Gene. 1989; 78: 309-321Crossref PubMed Scopus (79) Google Scholar). Specific amino acid residues involved in catalytic and regulatory functions of Pfk from Escherichia coli (6Evans P.R. Hudson P.J. Nature. 1979; 279: 500-504Crossref PubMed Scopus (146) Google Scholar, 7Schirmer T. Evans P.R. Nature. 1990; 343: 140-145Crossref PubMed Scopus (234) Google Scholar) are conserved in yeast and mammalian Pfk genes. In eukaryotes the N-terminal half of a Pfk subunit obviously retained the catalytic function, whereas in the C-terminal half allosteric ligand binding sites have evolved from former catalytic and regulatory sites (4Poorman R.A. Randolph A. Kemp R.G. Heinrikson R.L. Nature. 1984; 309: 467-469Crossref PubMed Scopus (184) Google Scholar, 8Kemp R.G. Gunasekera D. Biochemistry. 2002; 41: 9426-9430Crossref PubMed Scopus (49) Google Scholar, 9Arvanitidis A. Heinisch J.J. J. Biol. Chem. 1994; 269: 8911-8918Abstract Full Text PDF PubMed Google Scholar). This assumption is supported by studies with mutants of Saccharomyces cerevisiae expressing only the α or the β subunit of Pfk. It was demonstrated that one subunit type alone is able to form an enzymatically active Pfk entity in vivo (10Heinisch J. Curr. Genet. 1986; 11: 227-234Crossref PubMed Scopus (37) Google Scholar, 11Klinder A. Kirchberger J. Edelmann A. Kopperschlaöger G. Yeast. 1998; 14: 323-334Crossref PubMed Scopus (16) Google Scholar). Crystallographic analysis showed that an active bacterial Pfk consists of four identical subunits (12Evans P.R. Farrants G.W. Hudson P.J. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1981; 293: 53-62Crossref PubMed Scopus (158) Google Scholar, 13Shirakihara Y. Evans P.R. J. Mol. Biol. 1988; 204: 973-994Crossref PubMed Scopus (246) Google Scholar). No high resolution structure of a eukaryotic Pfk is available yet. But electron microscopic studies with S. cerevisiae Pfk (ScPfk) at 10.8-Å resolution suggested an octameric enzyme assembly (14Ruiz T. Mechin I. Baör J. Rypniewski W. Kopperschlaöger G. Radermacher M. J. Struct. Biol. 2003; 143: 124-134Crossref PubMed Scopus (31) Google Scholar). Recently we co-purified a protein component together with the known Pfk α and β subunits (15Yuan W. Tuttle D.L. Shi Y.J. Ralph G.S. Dunn W.A. J. Cell Sci. 1997; 110: 1935-1945Crossref PubMed Google Scholar, 16Kirchberger J. Baör J. Schellenberger W. Dihazi H. Kopperschlaöger G. Yeast. 2002; 19: 933-947Crossref PubMed Scopus (8) Google Scholar, 17Edelmann A. Baör J. Yeast. 2002; 19: 949-956Crossref PubMed Scopus (6) Google Scholar) from the methylotrophic yeast Pichia pastoris. This unknown protein could only be separated under denaturating conditions and with loss of Pfk activity. Herein, we present the sequence of the co-purified component and describe this new protein as a constitutively bound and regulatory relevant subunit of Pfk from P. pastoris (PpPfk) and other Pichia sp. Based on the molecular mass of the native PpPfk and the molar ratio and the molecular mass of the individual subunits we propose an enzyme complex formed of four α, β, and γ subunits. Yeast Strains and Growth Conditions—Strains used for isolation of nucleic acids and for analysis of Pfk proteins are summarized in supplemental Table S1. Yeast cells were cultivated in YP medium (1% yeast extract and 2% BactoPepton) containing 2% glucose or 0.5% methanol at 30 °C under rotation up to the growth phase as indicated. Minimal medium containing 0.67% yeast nitrogen base, 0.5% ammonium sulfate, and 2% glucose supplemented with adenine and amino acids but lacking uracil was used as selective medium. All biochemicals for cell cultivation were purchased from Difco (BD Biosciences) and Invitrogen. Preparation of Cell-free Extract and Assays—Cell-free extract was prepared according to Schwock et al. (18Schwock J. Kirchberger J. Edelmann A. Kriegel T.M. Kopperschlaöger G. Yeast. 2004; 21: 483-494Crossref PubMed Scopus (11) Google Scholar). Pfk activity measurement followed basically procedures described elsewhere (16Kirchberger J. Baör J. Schellenberger W. Dihazi H. Kopperschlaöger G. Yeast. 2002; 19: 933-947Crossref PubMed Scopus (8) Google Scholar). For kinetic studies a Pfk assay with simultaneous ATP and Fru 6-P regeneration (100 mm imidazole/HCl, pH 6.6, 100 mm KCl, 10 mm MgCl2, 20 mm potassium phosphate, 0.2 mm NADH+, 0.6 mm phosphoenolpyruvate, 8.5 units of pyruvate kinase/ml, 7 units of lactate dehydrogenase/ml, 1 unit of fructose-1,6-bisphosphatase/ml; ATP, Fru 6-P, AMP, and Fru 2,6-P2 as indicated) was used (16Kirchberger J. Baör J. Schellenberger W. Dihazi H. Kopperschlaöger G. Yeast. 2002; 19: 933-947Crossref PubMed Scopus (8) Google Scholar). A two-state Monod-Wyman-Changeux model was applied to describe the ATP velocity curves under the assumptions: 1) an octameric allosteric mode, 2) AMP and Fru 2,6-P2 binding to the R-state enzyme only, and 3) ATP serves as substrate ( KSATP) in a hyperbolic manner, but acts also as allosteric inhibitor ( KTATP), V=V×[ATP](KSATP+[ATP])×11+L L=(m0×(1+[ATP]/KTATP)(1+[AMP]/KRAMP)×(1+[Fru2,6−P2]/KRFru2,6−P2)) where V is maximum activity, m0 is the allosteric constant, KSATP is the ATP Michaelis constant, KTATP is the ATP-binding constant of the T-state enzyme, and KRAMP and KRFru2,6−P2 are AMP and Fru 2,6-P2 binding constants of the R-state enzyme. For description of the Fru 6-P velocity curves and the dependence of Pfk activity on AMP and Fru 2,6-P2 concentrations, a generalized Hill equation was used, V=V0+(Vmax−V0)×([X]/KAXjbcnH(1+([X]/KAXjbcnH where X is Fru 6-P, AMP or Fru 2,6-P2, and KXA is the half-activity constant. The kinetic data were fitted to Equations 1-3 by non-linear regression analysis applying SigmaPlot 9.0 (Systat Software, Inc., San Jose, CA) that uses the Marquardt-Levenberg algorithm for minimization. Alcohol oxidase activity was measured in a reaction coupled to horseradish peroxidase and the oxidation of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich) in 100 mm potassium phosphate buffer, pH 7.5, at 25 °C according to the company's technical information. For determination of protein concentrations, the procedure of Bradford (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) was applied using bovine serum albumin as standard. Purification of Pfk from P. pastoris and Protein Sequencing—Pfk was isolated from cell-free extract of P. pastoris strain MH458 as described previously (16Kirchberger J. Baör J. Schellenberger W. Dihazi H. Kopperschlaöger G. Yeast. 2002; 19: 933-947Crossref PubMed Scopus (8) Google Scholar). N-terminal sequences of polypeptides were determined according to the Edman procedure using the Protein Sequencer 473A (Applied Biosystems, Foster City, CA). Tryptic in-gel digestion and MALDI mass spectrometry measurements of the generated tryptic peptides were carried out as described previously (20Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). The mass spectrometric measurements were performed on a Bruker Reflex MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with an ion gate and pulsed ion extraction. Post source decay fragment ion spectra were obtained by using the FAST method (Bruker Daltonik). Generation of a Polyclonal Antibody against the γ Subunit of PpPfk—Subunits of purified Pfk from P. pastoris strain MH458 were separated under reducing conditions by SDS-PAGE. The γ subunit was cut out, destained in 10% acetic acid containing 40% methanol at 4 °C, and extracted by electroelution (Electro-Eluter Model 422, Bio-Rad). Then, the protein was dialyzed against phosphate-buffered saline (PBS; 50 mm sodium phosphate, 150 mm NaCl, pH 7.0). 200 μg of antigen in complete Freund's adjuvant (0.5-ml final volume) was used for rabbit immunization. After 5 weeks the animal was boosted in the same way. Antiserum was fractionated by 50% ammonium sulfate saturation. The precipitated protein was dialyzed against 20 mm sodium phosphate buffer, pH 7.0, and loaded onto a protein-A-Sepharose CL-4B column (Amersham Biosciences). The antibody was eluted with 100 mm citrate buffer, pH 3.0, neutralized with 1 m Tris/HCl, pH 9.0, precipitated with ammonium sulfate, and dissolved in PBS. For affinity purification, purified PpPfk was covalently coupled with bromocyan-activated Sepharose 4B as recommended by the manufacturer (Amersham Biosciences). After washing with PBS, the antibody was eluted with 3 m MgCl2, dialyzed alternate against 155 mm NaCl and PBS, and stored at -20 °C. Protein concentration was calculated according to A1cm,1mg/ml279nm=1.35 (21Kirschenbaum D.M. Anal. Biochem. 1973; 56: 237-263Crossref PubMed Scopus (11) Google Scholar). Cloning of PpPFK3 Encoding the γ Subunit—Touchdown PCR was carried out with genomic DNA as template and HotStarTaq™ DNA Polymerase (Qiagen). Furthermore, degenerate primers 4 and 14 were used, which corresponded to the identified amino acid sequences of the γ subunit (all primers are listed in supplemental Table S2). PCR was performed under the following conditions: Predenaturation at 95 °C for 15 min was followed by cycles of denaturation at 94 °C for 30 s, annealing beginning at 72 °C for 30 s, and elongation at 72 °C for 90 s. The annealing temperature was lowered 1 °C per cycle to 50 °C, which then was applied for annealing in the next 20 cycles. To identify the 5′- and 3′-ends, rapid amplification of cDNA ends (RACE)-PCR was performed as described previously (17Edelmann A. Baör J. Yeast. 2002; 19: 949-956Crossref PubMed Scopus (6) Google Scholar) and according to the manufacturer's protocol (Gene-Racer™ kit with cloned avian myeloblastosis virus reverse transcriptase, Invitrogen). PCR fragments were subcloned into pCR2.1 (the TOPOT-mTA Cloning® kit for sequencing, Invitrogen) and sequenced in both directions using the ABI PRISM® Big-Dye™ Terminators version 2.0 Cycle Sequencing Kit (Applied Biosystems). Generation of Individual Pfk Subunit-deficient P. pastoris Strains—Pfk subunit-deficient P. pastoris strains were generated by homologous recombination. Each plasmid (pAE27, pAE28, and pAE34) harbored one of the PpPfk genes interrupted by URA3 from P. pastoris (plasmids are depicted in supplemental Fig. S1). Transformation of the P. pastoris strain JC307 his4 ura3 was performed by electroporation (P. pastoris adjustment, GenePulser Xcell, Bio-Rad). To screen mutants and to verify homologous recombination, Southern blot analyses were performed as described previously (22Edelmann A. Kruöger M. Schmid J. J. Clin. Microbiol. 2005; 43: 6164-6166Crossref PubMed Scopus (52) Google Scholar). SDS-PAGE/Western Blot Analysis—Western blot analysis followed the description of Baör et al. (23Baör J. Schellenberger J. Kopperschlaöger G. Yeast. 1997; 13: 1309-1317Crossref PubMed Google Scholar) with the exception of the use of 10% polyacrylamide gels. Polyclonal rabbit antibodies against the γ subunit of PpPfk (this work) and against the purified ScPfk (24Huse K. Kopperschlaöger G. FEBS Lett. 1983; 155: 50-54Crossref PubMed Scopus (8) Google Scholar) were applied. The anti-ScPfk antibody showed strong cross-reactivity to the α and β subunits of PpPfk. Immunological detection was performed with anti-rabbit-IgG peroxidase conjugate (Dianova, Germany) and a chemiluminescent detection ECL™ Western blotting system (Amersham Biosciences-GE Healthcare). FACS Analysis and Immunofluorescence Microscopy—Cells were characterized by FACS analysis using forward light scattering and side light scattering, reflecting cell size and cell complexity, respectively. These were recorded on linear scales. Flow cytometric analysis was performed using the FACSCalibur™ scanner equipped with CellQuest™ software (both BD Biosciences). Thus, cells were grown in YP medium containing 2% glucose to an optical density of A580 nm =∼10 and diluted 1:51 (v/v) in the respective medium. For analysis of the subcellular distribution of Pfk protein by immunofluorescence microscopy, P. pastoris cells (A580 nm ≈ 1) were processed according to Pringle et al. (25Pringle J.R. Adams A.E. Drubin D.G. Haarer B.K. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (601) Google Scholar). Polyclonal antibodies against Pfk subunits (see SDS-PAGE/Western blot analysis) were used for specific protein detection. Cy3-labeled goat anti-rabbit-IgG antibody (Dianova, Hamburg, Germany) 200-fold (v/v) diluted with PBS containing 0.1% bovine serum albumin was applied as secondary antibody. Nucleus staining was performed with 4′,6-diamidino-2-phenylindol (1 μg/ml in PBS, Serva, Heidelberg, Germany) at room temperature for 5 min. Fluorescence images were obtained with a fluorescence microscope (Leica DM 5000B, Leica Microsystems CMS GmbH, Wetzlar, Germany) equipped with a 63×/1.4-0.6 oil immersion objective, a DFC350FX camera, and FW4000 software. Immunoprecipitation of PpPfk—The polyclonal antibody against the γ subunit of PpPfk (44 μl of affinity-purified IgG fraction; 0.6 μg/μl) was mixed with 200 μl of cell-free extract and stored at 4 °C for 30 min. Then, 20 mg of protein-A-agarose (wet weight, Roche Applied Science) washed with PBS were added. Following incubation at 4 °C for 3 h and centrifugation at 14,000 × g for 1 min, the gel was washed twice with ice-cold PBS. Immunoprecipitated proteins were released by incubation with 20 μl of 65 mm Tris/HCl buffer, pH 6.8, containing 20% Bromphenol Blue, 20% glycerol, 5% 2-mercaptoethanol, and 2% SDS in a boiling water bath for 5 min. Protein samples were analyzed by SDS-PAGE and Western blotting. Molecular Identification of a Pfk γ Subunit in P. pastoris—The protein band corresponding to the unknown polypeptide chain, which was co-purified together with the α and β subunits of PpPfk, was isolated from SDS-PAGE gel. Then, the N-terminal amino acid sequences of this protein and of several fragments obtained by chymotrypsin or trypsin degradation were determined by Edman procedure and MALDI-TOF post source decay analysis. Amino acid sequences identified are summarized in Table 1. Based on these results, degenerate primers were designed (supplemental Table S2). PCR and cloning techniques (see "Experimental Procedures") revealed a genomic DNA fragment of 3113 bp containing a complete coding sequence of 1056 bp (GenBank™ accession number AY686600). The transcription start was found at -43 bp from start ATG by 5′-RACE-PCR from mRNA. The coding sequence, further referred to as PpPFK3 (according to the nomenclature of other Pfk genes), encodes a polypeptide with a predicted molecular mass of 40.8 kDa (Fig. 1). N-terminal and internal amino acid sequences of the co-purified component determined by protein sequencing were identical to the respective sequence regions of the translated open reading frame of PpPFK3. However, the potential γ subunit appears at ∼34 kDa when purified PpPfk is analyzed by SDS-PAGE (see Fig. 5). The integrity of the N- and C-terminal ends of the γ subunit isolated by SDS-PAGE (Table 1) was verified by Edman and MALDI-TOF analyses, respectively. Therefore, proteolytic modifications of this protein can be excluded. Accordingly, the discrepancy between the sequence-based calculated mass and the apparent molecular mass found by SDS-PAGE is caused by the specific migration property of the γ subunit in this electrophoresis.TABLE 1Protein sequence analysis of the γ subunit of PpPfkFragmentMolecular mass, mono[M + H]+Putative sequenceaNumbers in parentheses represent amino acid position.DaEDMAN sequencingbPurified PpPfk from P. pastoris strain MH458 was subjected to SDS-PAGE. The γ subunit, co-purified with the α and β subunits, was excised and partially sequenced by Edman and MALDI-TOF post source decay techniques.(M)VTKDSIIRDLERENVGPEFGEFLNTLQTDLNSN terminus γ subunit (1-33)RSSR[PW]EDKVKGPALAN terminus internal sequence (limited proteolysis, γ') (163-177)MALDI-TOF post source decay analysis (sequencing)bPurified PpPfk from P. pastoris strain MH458 was subjected to SDS-PAGE. The γ subunit, co-purified with the α and β subunits, was excised and partially sequenced by Edman and MALDI-TOF post source decay techniques.YSDFVR785.37Internal sequence (134-139)SFVT[LI][LI][LI]DYY[QK]R1516.79/1516.83Internal sequence (222-233)MALDI-TOF analysiscIn-gel tryptic digestion and MALDI-TOF analysis were used to verify the integrity of the C-terminal part of the γ subunit.RHEIANFLK1127.75Internal sequence (301-309)SFVTLLLDYYQR1517.39Internal sequence (222-233)FHQGNISIHQISGYLD1829.03C-terminal sequence (336-351)KFHQGNISIHQISGYLD1956.34C-terminal sequence (335-351)IDLLLLTNNFDTNMNNK1993.19Internal sequence (240-256)MILVGDDRETDFEMSDR2029.21Internal sequence (205-221)QDLPLDYYLVLNNSQTGK2080.79Internal sequence (116-133)KIDLLLLTNNFDTNMNNK2121.27Internal sequence (239-256)SQLETHFNLAHETQEFSR2174.66Internal sequence (43-60)DLSIPLNVWFVLDMISQLSTSK2506.78Internal sequence (94-115)YLIYEAVGAEIHCFEQGSMPEQYR2833.45Internal sequence (140-163)QDLPLDYYLVLNNSQTGKYSDFVR2849.04Internal sequence (116-139)LLTNYYNNYEVNVLEFVLQMGFSR2927.32Internal sequence (70-93)a Numbers in parentheses represent amino acid position.b Purified PpPfk from P. pastoris strain MH458 was subjected to SDS-PAGE. The γ subunit, co-purified with the α and β subunits, was excised and partially sequenced by Edman and MALDI-TOF post source decay techniques.c In-gel tryptic digestion and MALDI-TOF analysis were used to verify the integrity of the C-terminal part of the γ subunit. Open table in a new tab FIGURE 5Limited proteolysis of purified PpPfk. Purified PpPfk (150 μg) was dissolved in 50 mm sodium phosphate buffer containing 5 mm ATP (200 μl and pH 7.0). 2 μl of α-chymotrypsin (0.2 mg/ml) were added, and the sample was incubated at 25 °C for 2 h. Proteolytic activity was stopped by addition of phenylmethylsulfonyl fluoride (1 mm final concentration), and the sample was divided into two aliquots. One aliquot was kept for SDS-PAGE, and the other aliquot was subjected to gel filtration on an SE-HPLC BioSelect SEC400 column (Bio-Rad) using 50 mm sodium phosphate buffer, pH 7.0, containing 150 mm NaCl. Gel filtration yielded a main protein fraction of ∼900 kDa. This fraction was concentrated and analyzed by SDS-PAGE (lane 4) together with the non-size-fractionated chymotrypsin-modified PpPfk (lane 3) and the undigested PpPfk (lane 2). Precision Plus Protein Unstained Standard (Bio-Rad) was used as molecular mass standard. Proteins were stained with Coo-massie Blue R250.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Extensive sequence analyses with various bioinformatic tools (NCBI Blast analyses, Predict-Protein, ELM) revealed no significant sequence similarity to any sequence deposit in GenBank™ (last analysis February, 27, 2007). Within the cloned genomic 5′ non-coding region (∼2000 bp) we identified putative consensus sequences for GCR1. This transcription factor is involved in specific regulatory mechanisms for glycolytic gene expression (26Clifton D. Weinstock S.B. Fraenkel D.G. Genetics. 1978; 88: 1-11Crossref PubMed Google Scholar, 27Holland M.J. Yokoi T. Holland J.P. Myambo K. Innis M.A. Mol. Cell. Biol. 1987; 7: 813-820Crossref PubMed Scopus (60) Google Scholar). In addition, an incomplete open reading frame of a hypothetical protein was detected. It shows sequence homology to the hypothetical proteins CAHO2429.1 and DEHAOCO3872.g from Kluyveromyces lactis (8/324 = 27%) and from Debaryomyces hansenii CBS767 (111/311 = 35%), respectively. Screening Other Yeasts for the Presence of the Pfk γ Subunit—To address the question whether the γ subunit is unique to P. pastoris, several other yeasts (supplemental Table S1) were initially screened for immuno-cross-reactivity by Western blot analysis. For this purpose, we used the polyclonal antibody against the PpPfk γ subunit. Whereas different P. pastoris strains and several other Pichia species displayed an immunoreactive band between 34 and 42 kDa, all extracts of distantly related yeasts (see supplemental Table S1) showed no specific immunoreactivity (data not shown). Next, PCR was applied to amplify ortholog sequences using degenerate primer sets designed on the basis of the γ subunit sequence from P. pastoris strain MH458. So far, the presence of a γ subunit was verified in P. pastoris strains JC307 and GS115, and in P. pseudopastoris (GenBank™ accession numbers DQ352840, DQ374390, and DQ386148). The γ subunits from GS115 are identical and show 95.4% amino acid identity to the γ subunit of P. pastoris strain MH458. The γ subunit of P. pseudopastoris, a species closely related to P. pastoris (28Dlauchy D. Tornai-Lehoczki J. Fulop L. Peter G. Antonie Van. Leeuwenhoek. 2003; 83: 327-332Crossref PubMed Scopus (37) Google Scholar), displays 79.8% identity at the amino acid level to the γ subunits from P. pastoris strains. Generation and Functional Characterization of γ Subunit-deficient P. pastoris Strains—To analyze the relevance of the γ subunit, three P. pastoris mutants were generated by deletion of PpPFK3. Correct recombination and gene deletion were confirmed by Southern and Western blot analyses (supplemental Fig. S2). The transformed recipient P. pastoris strain, which contained the Ura3 marker gene homologously integrated at the endogenous ura3 locus but still maintained an intact PpPFK3 locus, served as proper control (further referred to as wild-type strain). First, basic cell functions of the three γ subunit-deficient strains were studied. Since these strains behaved identically in all experiments, data are exemplarily shown for JC307-22ΔPpPFK3 (further referred to as γ subunit-deficient strain). Growth of the γ subunit-deficient strain was significantly reduced by 20% after 22 h of cultivation (Fig. 2A). This effect was found also under cultivation in a continuously oxygenized atmosphere. Deletion of the γ subunit did not interfere with the specific Pfk activity in cell-free extracts (Fig. 2B). Wild-type and γ subunit-deficient strains were able to grow on medium containing glycerol, rhamnose, or cycloheximide. This was determined by cultivation on pre-made culture plates (ID32C bioMerieux system) at 30 °C for 4 days (data not shown). Temperature sensitivity and NaCl tolerance were also indistinguishable between the strains (supplemental Fig. S3). Further, we analyzed the adaptation ability of the mutant and wild-type strains to glucose-containing medium after cultivation on methanol. In both cases a 50% reduction of alcohol oxidase activity over 3 h was observed (supplemental Fig. S4A) as a result of induced degradation of peroxisomes (microautophagy) (15Yuan W. Tuttle D.L. Shi Y.J. Ralph G.S. Dunn W.A. J. Cell Sci. 1997; 110: 1935-1945Crossref PubMed Google Scholar, 29Sakai Y. Koller A. Rangell L.K. Keller G.A. Subramani S. J. Cell Biol. 1998; 141: 625-636Crossref PubMed Scopus (197) Google Scholar). This was accompanied by a 50% increase in Pfk activity (supplemental Fig. S4B). Further, the strains were tested for endocytosis and vacuolar morphology by in vivo labeling (30Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1141) Google Scholar). For this purpose we used the fluorescent lipophilic dye FM1-43 (Molecular probes, Brussels, Belgium). FM1-43 internalization from the plasma membrane into endosomes and vacuolar membranes was indistinguishable between mutant and wild-type strains (supplemental Fig. S5). Although most of the phenotypes of the wild-type and the γ subunit-deficient strains were very similar, cells of the wild-type strain show remarkable flocculation with increasing cell density. The cells started to adhere at the middle log growth phase. The formed macroscopic flocs rapidly sedimented when continuous shaking was stopped. This phenotype was greatly reduced in γ subunit-deficient strains (Fig. 3). We observed that cell-cell adhesion in the wild-type strain was abolished in the presence of 2 mm EDTA but occurred again after the addition of 5 mm Ca2+. Adhesion of cells lacking the γ subunit was also induced by addition of Ca2+ but to a lesser extent (data not shown). Differences in the cellular structure of the γ subunit-deficient and the wild-type strains were also reflected by FACS analysis (supplemental Fig. S6). Kinetic Properties of the Wild-type and γ Subunit-deficient PpPfks—Because the γ subunit is not essential for the catalytic function of PpPfk per se (see above), we initiated an in-depth kinetic analysis of PpPfks purified from wild-type and γ subunit-deficient strains (Tables 2 and 3).TABLE 2Comparison of kinetic properties of purified Pfks from P. pastoris wild type and γ subunit-deficient strainsParameterPpPfkPpPfkWild typeγ-DeficientWithout effectoraMeasurement was carried out at 3 mm ATP.KFru 6-P0.5 (mm)2.17 ± 0.142.29 ± 0.18nFru 6-PH (mm)2.30 ± 0.102.00 ± 0.10Plus 1 mm AMPaMeasurement was carried out at 3 mm ATP.KFru 6-P0.5 (mm)0.17 ± 0.030.23 ± 0.03nFru 6-PH (mm)1.30 ± 0.101.10 ± 0.10Plus 20 μm Fru 2,6-P2aMeasurement was carried out at 3 mm ATP.KFru 6-P0.5 (mm)0.28 ± 0.020.33 ± 0.03nFru 6-PH (mm)1.20 ± 0.101.00 ± 0.10Half-activation constants of allosteric effectorsbMeasurement was carried out at 0.3 mm Fru 6-P and 3 mM ATP.KFru 2,6-P2A (μm)16.10 ± 1.1014.10 ± 1.00nFru 2,6-P2H (μm)1.20 ± 0.101.00 ± 0.10KAMPA (mm)0.12 ± 0.010.14 ± 0.02nAMPH (mm)1.60 ± 0.101.20 ± 0.10Inhibition by 5 mm ATPcData were obtained by measuring at 0.3 mm Fru 6-P without allosteric activator.Residual Pfk activity<0.05<0.05a Measurement was carr
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