New Results

Mitochondrial targeting of glycolysis in a major lineage of eukaryotes

Carolina Río Bártulos, Matthew B. Rogers, Tom A. Williams, Eleni Gentekaki, Henner Brinkmann, Rüdiger Cerff, Marie-Françoise Liaud, Adrian B. Hehl, Nigel R. Yarlett, Ansgar Gruber, Peter G. Kroth, Mark van der Giezen
doi: https://doi.org/10.1101/257790

Abstract

Glycolysis is a major cytosolic catabolic pathway that provides ATP for many organisms1. Mitochondria play an even more important role in the provision of additional cellular ATP for eukaryotes2. Here, we show that in many stramenopiles, the C3 part of glycolysis is localised in mitochondria. We discovered genuine mitochondrial targeting signals on the six last enzymes of glycolysis. These targeting signals are recognised and sufficient to import GFP into mitochondria of a heterologous host. Analysis of eukaryotic genomes identified these targeting signals on many glycolytic C3 enzymes in a large group of eukaryotes found in the SAR supergroup3, in particular the stramenopiles. Stramenopiles, or heterokonts, are a large group of ecologically important eukaryotes that includes multi- and unicellular algae such as kelp and diatoms, but also economically important oomycete pathogens such as Phytophthora infestans. Confocal immunomicroscopy confirmed the mitochondrial location of glycolytic enzymes for the human parasite Blastocystis. Enzyme assays on cellular fractions confirmed the presence of the C3 part of glycolysis in Blastocystis mitochondria. These activities are sensitive to treatment with proteases and Triton X-100 but not proteases alone. Our work clearly shows that core cellular metabolism is more plastic than previously imagined and suggests new strategies to combat stramenopile pathogens such as the causative agent of late potato blight, P. infestans.

Mitochondria provide the bulk of cellular ATP for eukaryotes by means of regenerating reduced NAD via the electron transport chain and oxidative phosphorylation2. In addition, mitochondria are essential for the production of iron-sulfur clusters4, and play roles in heme synthesis, fatty acid and amino acid metabolism5. Cytosolic pyruvate is decarboxylated by mitochondrial pyruvate dehydrogenase into acetyl-CoA which enters the citric acid cycle, subsequently producing one GTP (or ATP) and precursors for several anabolic pathways. More importantly, the reduction of NAD+ to NADH and production of succinate power the electron transport pathway and oxidative phosphorylation, being responsible for the majority of cellular ATP synthesis. The pyruvate is produced by glycolysis, a widespread cytosolic pathway that converts the six-carbon sugar glucose via a series of ten reactions into the three-carbon sugar pyruvate. Glycolysis is nearly universally present in the cytosol of most eukaryotes but also found in specialised microbodies known as glycosomes originally described in trypanosomatids6. More recently, two glycolytic enzymes were also found to be targeted to peroxisomes in fungi due to post-transcriptional processes7.

When analysing the genome of the intestinal parasite Blastocystis8, we discovered putative mitochondrial targeting signals on phosphoglycerate kinase (PGK) and on a fusion protein of triose phosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH). The amino-terminal sequences conform to typical mitochondrial targeting signals9 and are easily predicted by programmes such as MitoProt10. Analysis of the Blastocystis TPI-GAPDH and PGK sequences predicts a mitochondrial localisation with high probabilities (P 0.99 and 0.97, respectively). The predicted cleavage sites coincide with the start of the cytosolic enzymes from other organisms (Supplementary Fig. S1A) suggesting that these amino-terminal sequences might target both proteins to the mitochondrial organelle in this parasite11. We confirmed the functionality and sufficiency of these putative targeting signals by targeting GFP fused to these signals to mitochondria of a heterologous stramenopile host (Supplementary Fig. S2). Homologous antibodies were raised against Blastocystis TPI-GAPDH and PGK to test whether these proteins show an organellar localisation. Compartmentalised distribution of both TPI-GAPDH and PGK was clearly demonstrated using confocal microscopy and 3-dimensional rendering of optical sections (Fig. 1 A-D). Both proteins co-localise with the mitochondrial marker dye MitoTracker and in addition with DAPI which labels the organellar genomic DNA11,12 (Fig. 1 E-I).

Figure 1.
Figure 1.

The glycolytic enzymes TPI-GAPDH and PGK localize to mitochondria in the human parasite Blastocystis. Three-dimensional immunoconfocal microscopy reconstruction of optical sections (volume rendering) showing representative subcellular localization of PGK (blue) and TPI (red) in trophozoites (A-D). PGK (A) and TPI (B) volume signals show distinct distributions, consistent with localization within mitochondria, with considerable overlap. The merged image (C) provides a qualitative and the scatterplot (inset) of a quantitative measure of signal overlap. Co-localisation of MitoTracker (red) and PGK (green) and DAPI (blue) in trophozoites (E-I). Merged images MitoTracker/DAPI (E) PGK/DAPI (F) and TPI (G) and all three markers together (H) show considerable overlap. Scatterplots (inset) give a quantitative measure of signal overlap for each merged pair of markers (E-G). The DAPI signals (blue) representing nuclear DNA are indicated by asterisks (E). Scale bar 3 μm (A-D) or 2 μm (E-I).

The unexpected mitochondrial localisation of three glycolytic enzymes in Blastocystis prompted the analysis of all glycolytic enzymes in this intestinal parasite. Interestingly, targeting signals were only observed on the enzymes of the pay-off phase of glycolysis but not the investment phase (Fig. 2). Although three-dimensional reconstruction of our confocal microscopy data strongly indicated that these enzymes are indeed localised inside Blastocystis mitochondria (Fig. 1), we decided to confirm these findings using classical enzyme assays following cellular fractionation. These assays clearly showed that five C3 enzymes are found in the mitochondrial pellet while the five upstream enzymes are all confined to the soluble fraction (Table 1). To assess whether the putative mitochondrial enzymes were only laterally attached to the organelles, as in the case of hexokinase to VDAC in tumours1, we tested the latency of enzymatic activities in the presence or absence of Triton X-100. The increase of measurable activity of the C3 enzymes (not shown) suggests they are retained within a membranous compartment. The addition of proteolytic enzymes only affected the measured activity in the presence of the detergent Triton X-100 (Supplementary Table S1), clearly demonstrating that the five C3 glycolytic enzymes in Blastocystis are protected by a membrane and reside inside the mitochondria and not on the outside of the organelle, as observed in certain tumours1 or as in some proteomics studies13,14.

Figure 2.
Figure 2.

Stramenopile glycolytic enzymes contain mitochondrial-like amino-terminal targeting sequences. Representative stramenopiles with whole genome data known are shown. Presence of mitochondrial-like targeting signal is shown with a filled circle while open circle indicates no mitochondrial-like targeting signal. Where multiple isoforms with and without targeting signal exist, a half-filled circle is shown.

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Table 1.

Pay-off phase glycolytic enzymes in Blastocystis are found in the pellet. Activities of glycolytic enzymes from whole cell free extracts (c.f.e.) of Blastocystis suspended in phosphate buffered isotonic sucrose solution (pH 7.2). Cells were mixed at a ratio of two volumes of cells: three volumes of 0.5 mm glass beads and broken by three shakes of one minute each at maximum speed on a bead beater (VWR mini bead mill homogenizer (Atlanta, GA, USA)). Cell-free extracts were subjected to increasing centrifugal force producing nuclear, mitochondrial (pellet), lysosomal and cytosolic (supernatant) fractions at 1,912 RCFav for 5 min, 6,723 RCFav for 15 min, 26,892 RCFav for 30 min, respectively. Enzyme activities are the average of three determinations + SD. *1 enzyme unit (EU) is the amount of enzyme that converts 1 μmole substrate to product per minute. The yellow box indicates the site of major activity (or in the case of triosephosphate isomerase, the dual localization).

As some of us previously reported the mitochondrial localisation of the TPI-GAPDH fusion protein in a related stramenopile15, we wondered whether mitochondrial targeting of glycolytic enzymes is more widespread in this group of organisms. When querying all available stramenopile genomes, we noticed the widespread presence of mitochondrial targeting signals on glycolytic enzymes within the whole group. Here, as with Blastocystis, only enzymes of the C3 part of glycolysis seem to contain mitochondrial targeting signals (Fig. 2 and supplementary Fig. S1B). To test for functionality, all mitochondrial targeting signals from Phaeodactylum C3 glycolytic enzymes were fused to GFP and their cellular location was determined (Supplementary Fig. S2). As with Blastocystis, all constructs were targeted to the mitochondrion suggesting these are genuine mitochondrial targeting signals in vivo. In addition, we tested mitochondrial targeting signals found on glycolytic enzymes of the oomycete pathogen Phytophthora infestans, the water mould Achlya bisexualis and the multicellular brown alga Saccharina latissima, commonly known as kelp (Supplementary Fig. S2). In all cases, these targeting signals targeted GFP into mitochondria. However, some organisms also contain non-targeting signal bearing glycolytic enzymes suggesting that these cells likely have a branched glycolysis (see Supplementary Fig. S4 for P. tricornutum).

Previously, some bioinformatics studies16,17 had hinted at the possible mitochondrial location of several glycolytic enzymes. Here, using molecular, biochemical and cell biological methods, we clearly demonstrate the mitochondrial location of glycolytic enzymes of the pay-off phase of glycolysis in a major group of eukaryotes comprising both microbial and multicellular forms. The mitochondrial proteome has a complex and contested evolutionary past18,19, and we wondered if glycolytic enzymes targeted to mitochondria might have different evolutionary origins than those that operate in the cytosol. Phylogenetic analysis of all glycolytic enzymes provided no support for this hypothesis because stramenopile glycolytic enzymes cluster with the cytosolic forms of other eukaryotes in phylogenetic trees (Supplementary Figure S3 A-F). This result suggests that the canonical, cytosolic enzymes of glycolysis were targeted to the mitochondrion during stramenopile evolution.

It is difficult to conclusively determine the selective rationale, if any, for the retargeting of glycolysis to stramenopile mitochondria. In Blastocystis, and similar to many parasitic eukaryotes20, two key glycolytic enzymes have been replaced by pyrophosphate using versions. Normally, the reactions catalysed by phosphofructokinase and pyruvate kinase are virtually irreversible. However, the reactions performed by diphosphate-fructose-6-phosphate 1-phosphotransferase and phosphoenolpyruvate synthase (pyruvate, water dikinase) are reversible, due to the smaller free-energy change in the reaction. As Blastocystis is an anaerobe and does not contain normal mitochondrial oxidative phosphorylation11, any ATP not invested during glycolysis might be a selective advantage. However, in the absence of these irreversible control points there is a risk of uncontrolled glycolytic oscillations21. Separating the investment phase from the pay-off phase by the mitochondrial membrane might therefore prevent futile cycling. However, as not all stramenopiles use pyrophosphate enzymes, this cannot be the whole explanation.

The end-product of glycolysis, pyruvate, is transported into mitochondria via a specific mitochondrial transporter that has only recently been identified22 and that is absent from the Blastocystis genome8. The translocation of the C3 part of glycolysis into mitochondria would necessitate a novel transporter (presumably for triose phosphates). The identification and characterisation of such a transporter would open up new possible drug targets against important pathogens. Examples include Phytophthora infestans, the causative agent of late potato blight, which has a devastating effect on food security, but also fish parasites such as Saprolegnia parasitica and Aphanomyces invadans. Both have serious consequences for aquaculture and the latter causes epizootic ulcerative syndrome, an OIE listed disease23,24. Our recent genome analysis of Blastocystis identified several putative candidate transporters lacking clear homology to non-stramenopile organisms8. Such a unique transporter would not be present in the host (including humans) and could be exploited to prevent, or control, disease outbreaks that currently affect food production while the world population continuous to increase25.

Materials and methods

Sources of cDNA and genomic DNA

DNA and cDNA from Blastocystis ST1 strain NandII, obtained from a symptomatic human (strain obtained from the American Type Culture Collection, ATCC 50177), was used in this study. Genomic and cDNA libraries of Phaeodactylum tricornutum (culture from SAG strain: 1090-1a, Göttingen) were constructed with the "Lambda ZAP II XR library Construction Kit" from Stratagene and the lambda vector EMBL3, respectively. P. tricornutum Bohlin (strain 646; University of Texas Culture Collection, Austin) RNA was isolated using TRIzol following manufactures protocol (Thermo Fisher, Germany) and cDNA synthesis was performed with the reverse Transcription system (A3500, Promega, Germany). An Achlya bisexualis cDNA library1 was kindly provided by D. Bhattacharya (Rutgers University). Screening of libraries, sequencing of positive clones and RACE analyses were performed as described2. Phytophthora infestans RNA extracted from P. infestans mycelia with the RNAeasy Plant Kit from Quiagen and cDNA was synthesized with the Thermo-RT Kit (Display Systems, England). Sequences were also obtained from the EST/genome sequencing programmes from Phaeodactylum tricornutum3 and http://genome.jgi-psf.org/Phatr2/Phatr2.home.html(JGI)4, from Phytophthora infestans (http://www.pfgd.org5) and from P. sojae and P. ramorum (http://www.jgi.doe.gov6).

GFP constructs for the stable transformation of Phaeodactylum tricornutum

Standard cloning procedures were applied7. Polymerase chain reaction (PCR) was performed with a Master Cycler Gradient (Eppendorf) using Taq DNA Polymerase (Q BIOgene) according to the manufacturer’s instructions. cDNA from Blastocystis ST1 strain NandII (Bl), Phaeodactylum tricornutum (Pt), Phytophthora infestans (Pi) and Achlya bisexualis (Ab) was used as template for the PCR reactions. For Saccharina latissima (Sl) a cDNA clone (ABU96661) was used as template.

PCR products were cloned into TA-vector PCR 2.1 (Invitrogen) or blunt cloned into pBluescript II SK+ (Stratagene). The primers (Table 1) allowed insertion of restriction enzyme recognition sites (EcoRI/NcoI or SmaI/NcoI) that were used to clone the presequences in frame to eGFP within pBluescript-GFP. The presequence-GFP fusions were cut out with appropriate restrictions enzymes (EcoRI/HindIII or SmaI/HindIII) and cloned into the Phaeodactylum tricornutum transformation vector pPha-T18,9, either into the corresponding sites or, in case of SmaI, into the EcoRV site. For the constructs with Protein ID (Fig. S4) a slightly different cloning approach was used. PCR with a proof reading Polymerase (Pfu or Kapa Hifi) were used to amplify corresponding fragments from cDNA. These fragments were cloned blunt end in a modified pPha-T1 Vector. These Vectors include an eGFP with a StuI or KspAI restriction site, allowing a one-step cloning procedure, with subsequent screening for the correct orientation of the fragment at the N-terminus of eGFP. The Blastocystis presequences were produced by kinasing the primers using T4 polynucleotide kinase using manufacturer’s procedures and subsequently annealing in a thermal cycler after which they were cloned into the diatom expression vector equipped with eGFP and the StuI restriction site.

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Table 1:

PCR primers for production of GFP fusion constructs.

Transformation of Phaeodactylum tricornutum

Phaeodactylum tricornutum Bohlin (UTEX, strain 646) was grown at 22 °C under continuously light of 75 μE in artificial seawater (Tropic Marin) at a Q.5 concentration. Transformations were performed as described by Zaslavskaia et al.8,10 For each transformation, tungsten particles. For each transformation, tungsten particles M1Q (Q.7 μm median diameter) covered with 7-20 μg DNA were used to bombard cells with the Particle Delivery System PDS-1000 (Bio-Rad, HE-System) prepared with 650, 900, 1100 or 1350 psi rupture discs.

Microscopic analysis of transformed Phaeodactylum tricornutum

Reporter gene expression was visualized using confocal laser scanning microscopy (cLSM-510META, Carl Zeiss, Jena, Germany) using a Plan-Neofluar 40x/1.3 Oil DIC objective. The eGFP fusion proteins were excited with an argon laser at 488 nm with 8-10% of laser capacity. Excited fluorophores were detected with a bandpass filter GFP (505-530 nm) using a photomultiplier. Chlorophyll a autofluorescence was simultaneously detected with a META-channel (644-719 nm). MitoTraker Orange CM-H2TMRos (Molecular Probes) was applied for fluorescence staining of mitochondria. P. tricornutum cells were stained according to the protocol of the manufacturer. Cells were incubated with 100 nM dye solution, incubated for 30 minutes, washed and observed (images were recorded using the Multitracking mode with the following parameters for Wavelength T1 = 488 nm 10% and T2 = 543 nm 100% laser line, primary beam splitting mirrors UV/488/543/633 nm; emitted light was detected with the META-channel).

Protein production and antibody generation

Blastocystis TPI-GAPDH was amplified from cDNA using primers TPI-GAPDH pET F: aga aga CAT ATG TTC GTC GGT GGC AAT TGG AAG TGC AA and TPI-GAPDH pET R: tct tct GGA TCC TTA AGA GCG ATC CAC CTT CGC CA adding NdeI and BamHI restriction sites, respectively, to facilitate cloning in gene expression vector pET14b (Novagen, Merck, Whatford, UK). The Blastocystis PGK was amplified from cDNA using PGK pET F: aga aga CAT ATG AAG CTG GGA GTT GCT GCC TAC G and PGK pET R: tct tct CAT ATG TCA CGC GTC CGT CAG AGC GGC CAC ACC C which added NdeI restriction sites for pET14b cloning. The mitochondrial targeting signals were not amplified as these would not be part of the mature processed protein. All constructs were confirmed by sequencing. The in-frame His-tag allowed for affinity chromatography purification of the recombinant protein. Recombinant Blastocystis TPI-GAPDH and PGK were used to immunise guinea pigs and rabbits, respectively, for polyclonal antibody generation at Eurogentec (Seraing, Belgium).

Culture conditions for Blastocystis

Blastocystis isolate B (originally designated Blastocystis sp. group VII11, now called ST712) was used. The parasite was grown in 10 ml pre-reduced Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat-inactivated horse serum. Cultures were incubated for 48 h in anaerobic jars using an Oxoid AneroGen pack at 37 oC. Two-day-old cultures were centrifuged at 1600 g for 10 min, washed once in a buffer consisting of 30 mM potassium phosphate, 74 mM NaCl, 0.6 mM CaCl2 and 1.6 mM KCl, pH 7.4 and resuspended in an a nitrogen gassed isotonic buffer consisting of 200 mM sucrose (pH 7.2) containing 30 mM phosphate, 15 mM mercaptoethanol, 30 mM NaCl, 0.6 mM CaCl2, and 0.6 mM KCl (pH 7.2).

Subcellular fractionation of Blastocystis

Blastocystis cells were broken by mixing 2 volumes of the cell suspension with 3 volumes of 0.5 mm beads and broken by 3 one minute duration shakes at maximum speed on a bead breaker (VWR mini bead mill homogenizer, Atlanta, GA, USA) with one-minute pauses on ice. Cell-free extracts were subjected to increasing centrifugal force producing nuclear (N, 1,912 RCFav for 5 min), mitochondrialike (ML, 6,723 RCFav for 15 min), lysosomal (L, 26,892 RCFav for 30 min) and cytosolic (S) fractions, respectively, using a using a Sorvall RC-2B centrifuge fitted with an SS-34 rotor.

Enzyme assays

Hexokinase was assayed by measuring the reduction of NAD+ at 340 nm in a coupled reaction with Leuconostoc mesenteroides glucose-6-phophate dehydrogenase (3 EU), containing 38 mM Tris-HCl pH 7.6, 115 mM D-glucose, 10 mM MgCl2, 0.5 mM ATP, 0.2 mM NAD+, 0.05 mL of Blastocystis cell-free extract (0.08-0.12 mg) or fraction (N, 0.15-0.18 mg; ML, 0.12-0.17 mg; L, 0.08-0.11 mg; S, 0.090.05 mg), in a final volume of 1 mL at 25 oC.

Phosphoglucose isomerase was assayed by measuring contained g the reduction of NADP+ at 340 nm in a coupled reaction with Leuconostoc mesenteroides glucose-6-phophate dehydrogenase (2 EU), containing 38 mM Tris-HCl pH 7.6, 3.3 mM D-fructose-6-phosphate, 0.66 mM ß-NADP+, 3.3 mM MgCl2, 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Phosphofructokinase was assayed using the standard coupled assay containing 38 mM Tris-HCl pH 7.6, 5 mM dithiothreitol, 5 mM MgCl2, 0.28 mM NADH, 0.1 mM ATP, 0.1 mM AMP, 0.8 mM fructose-6-ohosphate, 0.4 mM (NH4)2SO4, 0.05 EU each of rabbit muscle aldolase, rabbit muscle glycerophosphate dehydrogenase, and rabbit muscle triosephosphate isomerase, 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Aldolase was assayed using a modification of the hydrazine method in which 3-phosphoglyceraldehyde reacts with hydrazine to form a hydrazone which absorbs at 240 nm; the assay contained 12 mM fructose-1,6-bisphosphate, pH 7.6, 0.1 mM EDTA, 3.5 mM hydrazine sulfate and 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Triosephosphate isomerase was assayed by measuring the oxidation of NADH using a linked reaction with glycerol-3-phosphate dehydrogenase; 220 mM triethanolamine pH 7.6, 0.20 mM DL-glyceraldehyde-3-phosphate, 0.27 mM NADH, 1.7 EU glycerol-3-phosphate dehydrogenase, and 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Glyceraldehyde-3-phosphate dehydrogenase was assayed by measuring the initial reduction of NAD+ at 340 nm; the assay contained 13 mM sodium pyrophosphate pH 8.0, 26 mM sodium arsenate, 0.25 mM NAD, 3.3 mM dithiothreitol, and 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Phosphoglycerate kinase was assayed by measuring the 3-phosphoglycerate dependent oxidation of NADH at 340 nm; the assay contained 40 mM Tris-HCl pH 8.0, 0.5 mM MgCl2, 0.26 mM NADH, 0.1 mM ATP, 2 EU S. cerevisiae glyceraldehydephosphate dehydrogenase, and 0.05 mL of B. hominis cell free extract or fraction in a final volume of 3 mL at 25 oC.

Phosphoglycerate mutase was measured using the standard coupled assay and measuring the decrease in absorbance at 340 nm; the assay contained 76 mM triethanolamine pH 8.0, 7 mM D(-) 3-phosphoglyceric acid, 0.7 mM ADP, 1.4 mM 2,3-diphosphoglyceric acid, 0.16 mM NADH, 2.6 mM MgSO4, 100 mM KCl, 5 EU pyruvate kinase/8 EU lactate dehydrogenase from rabbit muscle, 5 EU rabbit muscle enolase, and 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Enolase was determined using the standard coupled assay and measuring the decrease in absorbance at 340 nm; the assay contained 80 mM triethanolamine pH 8.0, 1.8 mM D(+) 2-phospholycerate, 0.1 mM NADH, 25 mM MgSO4, 100 mM KCl, 1.3 mM ADP, 5 EU pyruvate kinase/8 EU lactate dehydrogenase from rabbit muscle, and 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25°C.

Pyruvate kinase was determined by measuring the oxidation of NADH at 340 nm using the following mixture, 45 mM imidazole-HCl pH 8.0, 1.5 mM ADP, 0.2 mM NADH, 1.5 mM phosphoenolpyruvate, 5 EU rabbit muscle lactate dehydrogenase, and 0.05 mL of Blastocystis cell-free extract or fraction in a final volume of 3 mL at 25 oC.

Pyruvate phosphate dikinase was assayed spectrophotometrically by measuring the oxidation of NADH at 340 nm in 3 mL cuvettes. The reaction contained HEPES buffer (pH 8.0), 6 mM MgSO4, 25 mM NH4Cl, 5 mM dithiothreitol, 0.1 mM disodium pyrophosphate, 0.25 mM AMP, 0.1 mM phosphoenolpyruvate, and 0.05-0.25 mg of Blastocystis cell-free extract or fraction. The rate of pyruvate production was determined by the addition of 2 U of lactate dehydrogenase and 0.25 mM NADH, and compared to controls with phosphoenolpyruvate but lacking AMP, and those containing AMP but lacking phosphoenolpyruvate. The concentration of AMP, pyrophosphate and phosphoenolpyruvate used in the assay was selected from preliminary assays using varying concentrations from 0.025-1.0 mM. The generation of ATP from AMP by pyruvate phosphate dikinase was confirmed by measuring the ATP formed using a luciferin/luciferse assay (Molecular Probes, In Vitrogen, Eugene, OR, USA). The assay was performed as described above but lacking lactate dehydrogenase and NADH, after varying times 0, 15, 30, 45 and 60 min 0.1 mL of the assay is removed and added to one well of a 96 well plate containing 0.1 mL of 0.25 |j,g firefly luciferase and 0.5 mM luciferin and the luminescence recorded using a Spectra Max M2 plate reader (Molecular Devices, Sunnyvale, CA).

The activity of pyrophosphate dependent phosphofructokinase* in the direction of fructose-1,6-bisphosphate formation (forward reaction) was determined in 1 mL assay volumes containing 0.1 M HEPES-HCl, pH 7.8; 20 mM fructose-6-phosphate; 2 mM Na pyrophosphate; 5 mM MgCl2; 0.25 mM NADH; 0.2 U of aldolase (from rabbit muscle); and 0.3 U each of glycerophosphate dehydrogenase (from rabbit muscle) and triosephosphate isomerase (from rabbit muscle), 10 |J,M fructose 2,6 diphosphate. The reaction was initiated by addition of 0.05-0.25 mg of Blastocystis cell-free extract or fraction, and the rate of NADH oxidation was followed at 340 nm on a Beckman DU 640 spectrophotometer (Indianapolis, IN, USA). The activity of the reverse reaction was determined by measuring orthophosphate-dependent formation of fructose-6-phosphate from fructose-1,6-bisphosphate. The reaction mixture (1 mL) contained 0.1 M HEPES-HCl, pH 7.8; 2 mM fructose-1,6-bisphosphate; 15 mM NaH2PO4; 5 mM MgCl2; 0.3 mM NADP+ and 0.12 U glucose-6-phosphate dehydrogenase and 0.24 U glucose phosphate isomerase. The reaction was initiated by addition of 1 mg of pyrophosphate dependent phosphofructokinase and monitored at 340 nm. *Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP).

Confocal microscopy of Blastocystis

Blastocystis trophozoites were treated with MitoTracker Red (Molecular Probes), washed, fixed in 10% formalin and incubated in ice cold acetone for 15 minutes and air-dried.

Slides with fixed parasites were rehydrated in phosphate buffered saline (PBS) for 30 minutes and blocked with 2% BSA in PBS for 1 hour at room temperature. All antibody incubations were performed at room temperature in 2% BSA in PBS, 0.1% triton X-100. Slides were washed 5 times in 0.2 % BSA in PBS, 0.01% triton X-100 between incubations to remove unbound antibodies.

Primary antibodies: Rabbit, anti-PGK; Guinea Pig, anti-TPI-GAPDH (Eurogentec, Seraing, Belgium) were used at a dilution of 1:500 and 1:300 in 2% BSA in PBS, 0.1% triton X-100, respectively.

Secondary antibodies: Alexa Fluor 488 conjugated Goat anti-Rabbit (Invitrogen, Eugene, OR, USA), Alexa Fluor 405 conjugated Goat anti-Rabbit (Invitrogen, Eugene, OR, USA), TRITC-conjugated Goat anti-Guinea Pig were used at 1:200 dilutions in 2% BSA in PBS, 0.1% triton X-100, each.

The DNA intercalating agent 4’-6-Diamidino-2-phenylindole (DAPI) for detection of nuclear and mitochondrial DNA was added to the final but one washing solution at a concentration of 1 μg-ml−1. The labeled samples were embedded in Dako Glycergel Mounting Medium (DAKO, Carpinteira, CA, USA) and stored at 4 °C.

Immunofluorescence analysis and image data collection was performed on a Leica SP2 AOBS confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany) using a glycerol immersion objective lens (Leica, HCX PL APO CS 63x 1.3 Corr). Image z-stacks were collected with a pinhole setting of Airy 1 and twofold oversampling. Image stacks of optical sections were further processed using the Huygens deconvolution software package version 2.7 (Scientific Volume Imaging, Hilversum, NL). Three-dimensional reconstruction, volume and surface rendering, and quantification of signal overlap in the 3D volume model were generated with the Imaris software suite (Version 7.2.1, Bitplane, Zurich, Switzerland). The degree of signal overlap in the 3D volume model is depicted graphically as scatterplots. The intensity of two fluorescent signals in each voxel of the 3D model is measured and plotted. Voxels with similar signal intensity for both signals appear in the area of the diagonal. All image stacks were corrected for spectral shift before rendering and signal colocalization analysis.

Phylogenetic analyses

Sequences of all glycolytic enzymes from Phaeodactylum tricornutum and Blastocystis ST1, strain NandII, were used as seeds in BlastP searches in the non-redundant database at the NCBI13. We were especially interested to identify all sequences in the SAR supergroup14 (Stramenopiles, Alveolates and Rhizaria). In addition, representatives from other eukaryotic groups were added and, if required, closely related bacterial sequences. Sequences were automatically added to pre-existing alignments and subsequently manually refined using the Edit option of the MUST package15. Final datasets were generated after elimination of highly variable regions and positions with more than 50% gaps by G-blocks16. All datasets were first analysed with a maximum likelihood (ML) method under two different models. PhyML v2.317 was used with the SPR moves option and the LG+F+4G model18 and PhyML v3 (with SPR moves) was used using the C20+4G model, corresponding to 20 pre-calculated fixed profiles of positional amino-acid substitution18. Based on the likelihood values (l), the number of parameters (K) and alignment positions (n), the AIC (AIC= -2l +2K) and the corrected AIC (AICc; AIC+ 2K(K+1)/n-K-1) was calculated19. The lowest AICc value corresponds to the best tree, if the value of the C20 analysis was better, then a second ML analysis under the C40+4G model was performed and the AICc value estimated, until the overall best model was found. If the AICc of C40 is better than C20 then C60 was tested. Once the best model was estimated for all six datasets, a rapid bootstrap analysis with 100 replicates in RAxML v7 under the LG model was performed20 and an additional analysis in Phylobayes v3 with the CATfix C20 model in all cases or, alternatively, the best C-model. Two independent chains were run for 10,000 points and trees are sampled at every tenth points21. Trees obtained with the best model are presented and both posterior probabilities (PP) and rapid bootstrap values (BS) are indicated on trees if PP>0.5 or BS >30%, respectively.

Cellular localisation predictions

TargetP22 and MitoProt23 were used to analyse putative subcellular localization. Using the non-plant and no cut-offs settings. In case of Viridiplantae, Rhodophyta and Glaucocystophyta the plant results were taken, if non-plant results differ.

Amino acid sequences of mitochondrial targeting sequences used in GFP targeting experiments as seen in Supplementary Figure S2

A.

>preTPI-GAPDH-GFP (Blastocystis) (OAO12326) MLSRSSVIARSFGSAARKL >prePGK-GFP (Blastocystis) (OAO15536) MLSAFSKRLFSTGRTVN

B.

>preTPI-GAPDH-GFP (Phaeodactylum) (NCBI AF063804) MLASSRTAAASVQRMSSRAFHASSLTEARKFFVGGNWKCNGS >prePGK-GFP (Phaeodactylum)(JGI 48983) MLFRMLTSTALRRSPVTTSLTCCCKANAFAVRIRSFHAAPVIQAKMTVEQLAQQ >prePGM-GFP (Phaeodactylum)(JGI 33839) MFAVSRSSFLLATRVKTLRSFAAVQAADKHTLVLLRHGESTWNLENKFTGWYDCP >preENO-GFP (Phaeodactylum)(JGI 1572) MMWSRPVLRRNISTTRASSSSRRFLSAITGVHGREIDSRGNPTVEVDVTTAQGT >prePK-GFP (Phaeodactylum) (JGI 49002) MMRSFLRHAQGRACAQHLRTIGTLRLNQMPVTGA

C.

>preTPI-GAPDH-GFP (Phytophthora infestans) (NCBI X64537) MSFRQVFKTQARHMSSSSRKFFVGGNWKCNGSLGQAQELVGMLNTA >prePGM-GFP (Phytophthora infestans) (PfGD Pi_011_55705_Feb05.seq) MVLALRRPLAISSRVANRSLGMLRQQQKAMKHTHTLVLIRHGESEWNKKNLFTGWYDVQLSEKGNKEA >prePK-GFP (Achlya bisexualis) (NCBI AAU81895) MLARSLRSRAVRSFARGLSNKPSKNDAFSMT >preTPI-GAPDH-GFP (Saccharina latissima) (NCBI ABU96661) MFSAALSAAGAKAPSAARGFASSASRMSGRKFFVGGNWKCNGS

Phaeodactylum tricornutum amino acid sequences used in GFP targeting experiments as seen in Supplementary Figure S4

>preTPI_50738 plastid (pre-sequence) (JGI 50738) plastid MTGDSTSLLDLISPDRERPQRKEPSRWIAFSVFPFVRFIPEAFATRLPYSIVMKFLALSVAALISSATAFAPTFR GSPASTTASTTSLAARKPFISGNWKLN >preTPI_18228 plastid (pre-sequence) (JGI 18228) plastid MKFLALSVAALISSATAFAPTFRGSPASTTASTTSLAARKPFISGNWKLN >TPI_54738 cytosol (242 Amino acids) (JGI 54738) cytosol MPRPDGSSTPAAEGERKYLVAGNWKCNGTLASNEELVKTFNEAGPIPSNVEVAICCPSLYLPQLLSSLRDDIQIG AQDCGVNDKNGAFTGEIGAFQIKDIGCDWVIIGHSERRDGFEMPGETPDLCAKKTRVAIDAGLKVMFCIGEKKEQ REDGTTMDVCASQLEPLAAVLTESDWSSIAIAYEPVWAIGTGLTATPEMAQETHASIRDWISQNVSADVAGKVRI QYGGSMKGANAKDLLEQ >Gapdh3_23 598 cytosol (full length) (JGI 23598) cytosol MPVKCLVNGFGRIGRLCFRYAWDDPELEIVHVNDVCSCESAAYLVQYDSVHGTWSKSVVAAEDSQSFTVDGKLVT FSQEKDFTKIDFASLGVDMVMECTGKFLTVKTLQPYFGMGVKQVVVSAPVKEDGALNVVLGCNHQKLTTDHTLVT NASCTTNCLAPVVKVIQENFGIKHGCITTIHDVTGTQTLVDMPNTKKSDLRRARSGMTNLCPTSTGSATAIVEIY PELKGKLNGLAVRVPLLNASLTDCVFEVNKEVTVEEVNAALKKASESGPLKGILGYETKPLVSTDYTNDTRSSII DALSTQVIDKTMIKIYAWYDNEAGYSKRMAELCNIVAAMNITGQEPSFKYE >prePGK_29157 plastid (pre-sequence) (JGI 29157) plastid MKFVQAAIFALAASASTTAAFAPAKTFGVRSFAP >PGK_51125 cytosol (193 Amino acids) (JGI 51125) cytosol MASDMPKLAPGATRKRNVFDVIEALQKQSAKTILVRVDFNVPMNSDGKITDDSRIRGALPTIKAVVNAKCNAVLV SHMGRPKLVQKAADDEETRQQRHELSLKPVADHLAKLLDQEVLFGDDCLHAQSTIRELPAEGGGVCLLENLRFYK EEEKNGEDFRKTLASYADGYVNDAFGTSHRAHASVAGVPALLP >PGM_43812 unclear (130 Amino acids) (JGI 43812) unclear localization, cytosol plus ER or mitochondria MGRRTTHRRLFPALALIFAELIMSTAYSLAWRTSAACWTTTTGTACSRSRIATTRKVRRSRPNPCNPWHPVAFSF FGTSSRRCRSSGSLYGEIDADAEGPDSPSADDRSVPTPSTTSSLSRSETLPPIPP >PGM_43253 mitochondria (112 Amino acids) (JGI 43253) mitochondria MASITLNRSRFTMITAIGMSHPRSHGTPRSVLLLLLRQFSSKDWNSKGTDSASRSGPVLIKKTPRSAAAAKLRST APSLNGSTTDSTTGAVKHHPAHHYINGGTPCDPAPPP >PGM_26201 unclear (408 Amino acids) (JGI 26201) unclear, mitochondria or ER MLVPHPSGKAMRGLREEACRFLSSRSFGATLDATHARMGGNFVNSVQACNNGKRVCWHQRNRRTFSVVATQRNGI GHRTTQGETEAVPRRHFTSLNQSTPFQLCFLRHGQSTWNRDNIFIGWTDTPLTDDGVLEARVAGKMLHKSGIRFD EVHTSLLRRSIRTTNLALMELGQEYLPVHKHWRLNERCYGDLVGKNKKEVVMQHGADQVKRWRRSYDEPPPPMSD DHPYHPARDPRYQNILDELPKSESLKNTVERSSLYWDEVLAPALREGKTLLVVGHENNLRSLLMRLEDIAPEDII NLSLPRAVPLAYRLDENLKPLPREDGKLDEATGFLKGTWLGGDQAVSEILDRDHKQVYDTAITTNLEIGQDREKW NNWMEFIMGKPSAKQKRIGGDKQNGFAGGAAIP >PGM_42 8 57 plastid (175 Amino acids) (JGI 42857) plastid MAMDAITMRKLTLTMAVLLIVSGCEALLVFLPRRSPFTVISTRSSTNSAGLLHLHSKANESDGLEGKWIKVSSAL DEGVDAANEEKEGAFLSSDYNSMNGYNTDLNRYHTMLRERGTFVEALFGQRRSFVIAKRDGDENEDGWRDMRRQR RPLWKHLLRLPISVAKNVLWKPPQP >PGM_35164 mitochondria (351 Amino acids) (JGI 35164) mitochondria MRIPCRRLHPQLSAKGTRRPFQYSSSNSIDDQHRSSHLDASPGRHIVVRHGQSVWNKGSNQLERFTGWTNVGLSE NGQRQAVQAARKLHGYSIDCAYVSLLQRSQATLRLMLEELNDQGRRSEGYDDLTTDIPVISSWRLNERHYGALTG QSKLQAEQLFGKAQLDLWRYSYKIPPPPMDPDTFSSWKHQAHCQMATYIHHRHNRSRVIEKGNSVWDSSRAVMPR SEAFFDVLQRIVPLWKYGIAPRLARGETVLLVGHANSVKALLCLLDPHTVTPTSIGALKIPNTTPLVYQLIRDYP GASTSVPASFPVLGDLRVVIPPSNSTRYPLSGTWLEDPPVARDAGTAVEEP >PGM_51298 cytosol (131 Amino acids) (JGI 51298) cytosol. If a shorter version, starting from the second Methionine is used, a localization at the plastid as a blob like structure is the result (data not shown). MCDESRQTATPMIHFEIFRFSDPLVRQDRQAPHLSLTSTVKILSDSNLHKLFIMMLRSLVLALSWTVASAFTHQS TFWGRTAVTNSRILSLSPPTDASSSALCMKYMLVLVRHGESTWNKENRFTGWVDCP >preENO_56468 cytosol (66 Amino acids) (JGI56468) cytosol. Start Methionine from GFP was not included in the construct. MLFKPSTLLALFAVAGTTLAFAPRSTTTPLTSTTRGSASSSVTTLAMSGITGVLAREILDSRGNPV >ENO_56468 plastid (443 Amino acids) (JGI56468) plastid MLFKPSTLLALFAVAGTTLAFAPRSTTTPLTSTTRGSASSSVTTLAMSGITGVLAREILDSRGNPTVEVEVTTAD GVFRASVPSGASTDAYEAVELRDGGDRYMGKGVLQAVQNVNDILGPAVMGMDPVGQGSVDDVMLELDGTPNKANL GANAILGVSLAVAKAGAAAKKVPLYRHFADLAGNNLDTYTMPVPCFNVINGGSHAGNKLAFQEYFVIPTGAKSFA EAMQIGCEVYHTLGKIIKAKFGGDATLIGDEGGFAPPCDNREGCELIMEAISKAGYDGKCKIGLDVAASEFKVKG KDEYDLDFKYDGDIVSGEELGNLYQSLAADFPIVTIEDPFDEDDWENWSKFTTKNGATFQVVGDDLTVTNIEKIE RAIDEKACTCLLLKVNQIGSISESIAAVTKAKKAGWGVMTSHRSGETEDTYIADLAVGLCTGQIKTGA >PK_49098 cytosol (507 Amino acids) (JGI: 49098) cytosol MTASQTKITASGPELRGANITLDTIMKKTDVSTRQTKIVCTLGPACWEVEQLESLIDAGLSIARFNFSHGDHEGH KACLDRLRQAADHKKKHVAVMLDTKGPEIRSGFFADGAKKISLVKGETIVLTSDYSFKGDKHKLACSYPVLAKSV TPGQQILVADGSLVLTVLSCDEAAGEVSCRIENNAGIGERKNMNLPGVIVDLPTLTDKDIDDIQNWGIVNDIDFI AASFVRKASDVHKIREVLGEKGKGIKIICKIENQEGMDNYDEILEATDAIMVARGDLGMEIPPEKVFLAQKMMIR QANIAGKPVVTATQMLESMITNPRPTRAECSDVANAVLDGTDCVMLSGETANGEYPTAAVTIMSETCCEAEGAQN TNMLYQAVRNSTLSQYGILSTSESIASSAAKTAIDVGAKAIIVCSESGMTATQVAKFRPGRPIHVLTHDVRVARQ CSGYLRGASVEVISSMDQMDPAIDAYIERCKANGKAVAGDAFVVVTGTVAQRGVTNA >PK_56445 cytosol (538 Amino acids) (JGI 56445) cytosol MSLSQSSDVPILAGGFITLDTVKHPTNTINRRTKIVCTIGPACWNVDQLEILIESGMNVARFNFSHGDHAGHGAV LERVRQAAQNKGRNIAILLDTKGPEIRTGFFANGASKIELVKGETIVLTSDYKFKGDQHKLACSYPALAQSVTQG QQILVADGSLVLTVLQTDEAAGEVSCRIDNNASMGERKNMNLPGVKVDLPTFTEKDVDDIVNFGIKHKVDFIAAS FVRKQSDVANLRQLLAENGGQQIKICCKIENQEGLENYDEILQATDSIMVARGDLGMEIPPAKVFLAQKMMIREA NIAGKPVITATQMLESMINNPRPTRAECSDVANAVLDGTDCVMLSGETANGPYFEEAVKVMARTCCEAENSRNYN SLYSAVRSSVMAKYGSVPPEESLASSAVKTAIDVNARLILVLSESGMTAGYVSKFRPERAIVCLTPSDAVARQTG GILKGVHSYVVDNLDNTEELIAETGVEAVKAGIASVGDLMVVVSGTLYGIGKNNQVRVSVIEAPEGTVKETPAAM KRLVSFVYAADEI >PK_45997 cytosol (533 Amino acids) (JGI 45997) cytosol MLSSTSTIPKLDGEVVTLSIIKKPTETKKRRTKIICTLGPACWSEEGLGQLMDAGMNVARFNFSHGDHEGHGKVL ERLRKVAKEKKRNIAVLLDTKGPEIRTGFFADGIDKINLSKGDTIVLTTDYDFKGDSKRLACSYPTLAKSVTQGQ AILIADGSLVLTVLSIDTANNEVQCRVENNASIGERKNMNLPGVVVDLPTFTERDVNDIVNFGIKSKVDFIAASF VRKGSDVTNLRKLLADNGGPQIKIICKIENQEGLENYGDILEHTDAIMVARGDLGMEIPSSKVFLAQKYMIREAN VAGKPVVTATQMLESMVTNPRPTRAECSDVANAVYDGTDAVMLSGETANGPHFEKAVLVMARTCCEAESSRNYNL LFQSVRNSIVIARGGLSTGESMASSAVKSALDIEAKLIVVMSETGKMGNYVAKFRPGLSVLCMTPNETAARQASG LLLGMHTVVVDSLEKSEELVEELNYELVQSNFLKPGDKMVVIAGRMAGMKEQLRIVTLDEGKSYGHIVSGTSFFF ERTRLLDF >PK_56172 mitochondria (86 Amino acids) (JGI: 56172) mitochondria MFRRAVLSLSTRAIRTPVPCSVARGDASQVRSLAQTTFYLPDPADRSQDVHNRGNLQLSKIVATIGPTSEQEEPL RLVTDAGMRIM

Acknowledgements

The authors wish to thank Professors John F. Allen and Nick Lane (both UCL, UK) for fruitful discussions and criticism. Furthermore, we want to thank Ulrike Brand (TU-BS) and Doris Ballert (Uni KN) for technical assistance. TAW is supported by a Royal Society University Research Fellowship. Work in the lab of MvdG was supported by Wellcome Trust grant 078566/A/05/Z. PGK wishes to acknowledge support by the German Research Foundation (DFG, grant KR 1661/6-1), the Gordon and Betty Moore Foundation GBMF 4966 (grant DiaEdit), and the BioImaging Center of the University of Konstanz as well as Professor Mendel and his group (TU-BS) for using their equipment.

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Posted January 31, 2018.
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Mitochondrial targeting of glycolysis in a major lineage of eukaryotes
Carolina Río Bártulos, Matthew B. Rogers, Tom A. Williams, Eleni Gentekaki, Henner Brinkmann, Rüdiger Cerff, Marie-Françoise Liaud, Adrian B. Hehl, Nigel R. Yarlett, Ansgar Gruber, Peter G. Kroth, Mark van der Giezen
bioRxiv 257790; doi: https://doi.org/10.1101/257790
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