Comparative proteomics of glycosomes from bloodstream form and procyclic culture formTrypanosoma brucei brucei

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DOI 10.1002/pmic.200500668

RESEARCH ARTICLE

Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei Claudia Colasante, Margrit Ellis, Thomas Ruppert and Frank Voncken ZMBH, Heidelberg, Germany

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.

Received: September 15, 2005 Revised: January 24, 2006 Accepted: January 24, 2006

Keywords: Glycosome / Mass spectrometry / Metabolism / Peroxisome / Trypanosoma brucei brucei

1

Introduction

Peroxisomes are single membrane-bound organelles with a protein-dense matrix, which are present in most eukaryotic cells [1, 2]. They exhibit a broad functional versatility, and their enzyme content varies with both the organism in which they occur and the environmental conditions to which the organisms are exposed [2–7]. Medically and economically important Kinetoplastid parasites such as Trypanosoma sp. and Leishmania sp. contain an unusual peroxisome-like microbody, the glycosome, which contains the first seven to nine enzymes of glycolysis [4, 5, 8–12]. Correspondence: Dr. Frank Voncken, ZMBH, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany E-mail: [email protected] Fax: 149-6221-545894 Abbreviations: ADP, adenosinediphosphate; ATP, adenosinetriphosphate; BSF, bloodstream form; NAD, nicotinamide adenine dinucleotide; NADH, reduced form of NAD; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP; nanoLC-MS/MS, nano LC-ESI-Q-TOF-MS; PCF, procyclic culture form; PEP, phosphoenolpyruvate; PTS, peroxisomal targeting signal

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The African trypanosomes Trypanosoma brucei rhodesiense and T. b. gambiense cause sleeping sickness in humans, and T. b. brucei causes ‘nagana’ in cattle (http://www.who.int/tdr/ diseases). These trypanosomes possess a complex life cycle that includes two replicating stages: the bloodstream form (BSF) in the blood and tissue fluids of mammals, and the insect (procyclic) form (PCF) in the midgut of the vector, the tsetse fly [13, 14]. To survive in the two different hosts, considerable adaptations in parasite morphology, surface composition, and metabolism are required [14, 15]. The BSF of T. brucei sspp. primarily metabolize glucose to pyruvate and glycerol, and obtain adenosinetriphosphate (ATP) exclusively by substrate-level phosphorylation during glycolysis [11, 16]. The mitochondria of this life form are highly reduced and lack key enzymes and components of the Krebs cycle. Their role in energy metabolism is restricted to the re-oxidation of glycerol 3-phosphate by the mitochondrial alternative oxidase [11, 17]. This is in contrast to the PCF of T. brucei sspp., which have an elaborate mitochondrion where ATP is generated by a combination of substrate-level and oxidative phosphorylation [11, 18–20]. Substrates for the mitochondrial energy generation are products of the glycosomal glucose metabolism or amino acids like proline and threonine [11, 18–24]. www.proteomics-journal.com

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In addition to glycolysis, other pathways have been postulated to be present in the glycosome, like ether lipid biosynthesis, b-oxidation of fatty acids, sterol and isoprenoid biosynthesis, purine salvage, pyrimidine biosynthesis, the pentose phosphate pathway and gluconeogenesis [4, 5, 11]. The glycosomal compartmentalization of some of these enzymes and pathways was shown to be essential for trypanosome survival [25–27], and the glycosome has been long viewed as a possible drug target [28, 29]. Similar to peroxisomes [2], glycosomes were shown to contain peroxins (PEX proteins) that participate in the import of matrix proteins or the formation of a functional bounding membrane [27, 30– 32]. Identification of glycosomal constituents was mainly based on subcellular fractionation studies [4, 5, 8–11, 33]. Another indication was the presence of peroxisomal (glycosomal) targeting signals (PTS) in deduced protein sequences [5, 34, 35]. Some glycosomal matrix proteins, however, lack an identifiable PTS [36, 37], and there is no good algorithm for detecting glycosomal membrane proteins [38]. Any classification based on sequence information alone is therefore certain to be incomplete. Recently, MS-based organellar proteomics has been successfully applied to peroxisomes of fungi [39], plants [40], and mammals [41], and resulted in the identification of novel peroxisomal proteins. Here, we report a comparative (nonquantitative) proteomic analysis of glycosomes from both the BSF and the PCF of a well-characterized lab strain of T. b. brucei, which resulted in the validation of known functional aspects of glycosomes and in the identification of novel glycosomal constituents.

2

Materials and methods

2.1 Culture of trypanosomes T. b. brucei strain 449 BSF [42] was cultured in HMI-9 medium [43] and PCF in MEM-PROS medium [44], supplemented with 10% v/v fetal bovine serum (Sigma-Aldrich, Deisenhofen, Germany).

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many), and was ground in a pre-chilled mortar with one volume of wet-weight silicon carbide (Crystolon: Norton Company, Worcester MA: porous ,400 mesh). Using lightmicroscopy, the cells were checked for at least 90% disruption. The cell lysate was centrifuged at 1006g and 10006g, for 15 min each, to remove the abrasive and nuclei, respectively. The supernatant was centrifuged at 17 0006g for 15 min to yield the glycosome-enriched fraction, which was resuspended in 3 mL of homogenization buffer. A 32-mL linear 20–40% v/v Optiprep (Iodixanol: Axis-shield, Oslo, Norway) gradient, mounted on top of a 3.5-mL 50% v/v Optiprep cushion, was prepared according to the manufacturer’s protocol (Optiprep Application Sheet S9: available at http://www.axis-shield.com/density/dapp.htm). Centrifugation was performed at 170 0006g for 1 h at 47C using a Beckman VTi-50 Rotor (acceleration and deceleration 9). Aliquots of 1 mL were collected from the bottom of the tube after puncture, and the protein concentration of each fraction was determined using the Bradford assay (Bio-Rad, München, Germany). Equal volumes of each fraction were precipitated with TCA, and the protein pellets were resuspended in denaturing SDS-PAGE buffer [45]. Proteins were separated on a 12% SDS-PAGE gel and analyzed by Western blotting or stained with CBB R-250 (Sigma-Aldrich). 2.3 Western blotting For Western blotting, proteins were transferred to a Hybond PVDF membrane (Amersham Biosciences, Freiburg, Germany) in transfer buffer (39 mM glycine, 48 mM Tris-base, 0.037% SDS, pH 8.3) for 1 h at 100 V. The membranes were blocked in 7.5% non-fat dry milk in TBS/Tween 20 (0.2% v/v) for 1 h at room temperature with gentle shaking, and incubated with the appropriate primary antibodies for 1 h (see Fig. 1). The membranes were then washed once for 15 min, and twice for 5 min in TBS/Tween 20 (0.2% v/v), followed by incubation for 1 h at room temperature with the corresponding horseradish peroxidase-conjugated secondary antibodies. Finally, the membranes were washed once for 15 min, and twice for 5 min in PBS/Tween 20 (0.2% v/v), and developed according to the manual of the ECL detection kit (Amersham Biosciences).

2.2 Isolation of glycosomes BSF and PCF T. b. brucei were grown to an exponential-phase density of 16106 and 36106 cells/mL, respectively, and harvested by centrifugation for 10 min at 20006g to give approximately 361010 cells each. The cells were washed once in 50 mL of TEDS (25 mM Tris, 1 mM EDTA, 1 mM DTT, 250 mM sucrose pH 7.8). After centrifugation, the pellet was resuspended in TEDS/15% v/v glycerol, followed by a freeze (2807C)/thaw cycle. After centrifugation, the cell pellet was resuspended in 2 mL of homogenization medium (250 mM sucrose, 1 mM EDTA, 0.1% v/v ethanol, 5 mM MOPS, pH 7.2) supplemented with ‘Complete EDTA-Free’ protease-inhibitor cocktail (Roche Applied Science, Mannheim, Ger© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.4 In-gel trypsin digestion and nano LC-ESI-Q-TOF-MS analysis Proteins of the BSF and PCF glycosomal peak fractions number 8 were separated by SDS-PAGE and stained with CBB R250 (Sigma-Aldrich). Visible protein bands were excised with a scalpel, and the remaining gel lane was cut into pieces of approximately 3 mm each. Gel slices were transferred to a 96-well plate, reduced, alkylated and digested with trypsin [46] for 4 h using a Digest pro MS liquid handling system (Intavis, Köln, Germany). Following digestion, tryptic peptides were extracted from the gel pieces with a mixture of 50% ACN/0.1% TFA, concentrated nearly to drywww.proteomics-journal.com

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Figure 1. Protein concentrations and Western blotting of gradient fractions from (A) BSF and (B) PCF T. b. brucei. Upper panels: protein distribution profile; the protein concentration of each gradient fraction was determined and plotted against the fraction number. Lower panels: Western blot analysis using antibodies directed against marker proteins of different organelles. Glycosome-, mitochondrion-, lysosome-, and acidocalcisome-containing gradient fractions were identified with aPEX11 [30], aLPDH [141], aVP1L3 [142], and ap67 [143], respectively. (C) CBB R250 staining of total cell lysates (lanes 1) and the glycosome-enriched peak fractions 8 (lanes 2), isolated from BSF and PCF T. b. brucei. In each lane, 25 mg of protein was loaded.

ness in a speedVac vacuum centrifuge, and diluted to a total volume of 30 mL with 0.1% TFA. Of each sample, 25 mL was analyzed using a nanoHPLC system (UltiMate: Dionex, Amsterdam, The Netherlands), equipped with a FAMOS auto-sampler (Dionex), coupled to an ESI Q-TOF hybrid mass spectrometer (Applied Biosystems, Foster City, CA). Samples were loaded on an Inertsil C18-trapping column (LC Packings, Amsterdam, The Netherlands) using 0.1% TFA and a flow rate of 20 mL/min. Peptides were eluted and © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

separated on an analytical column (75 mm6150 mm) packed with Inertsil 3-mm C18 material (LC Packings) using a flow rate of 200 nL/min and applying a gradient of buffer A (0.1% formic acid/5% ACN) and buffer B (0.1% formic acid/80% ACN). The elution profile was as follows: 0–2 min, 5% B; 2– 50 min, 5–40% B; 50–60 min, 40–60% B; and 60–63 min, 60– 90% B. The column was connected to a nano-ESI emitter (New Objectives, Woburn, MA) and 2000 V were applied via liquid junction. The Q-TOF operated in positive ion mode. www.proteomics-journal.com

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One MS survey scan (0.7 s) was followed by one informationdependent product ion scan (3 s). Only double and triple charged ions were selected for fragmentation. 2.5 Identification of MS/MS spectra by database searches The MS/MS spectra were searched against the completed [47] genome database of T. b. brucei (http://www.tigr.org) using MASCOT software (Matrix Science, London, UK). The algorithm was set to: use trypsin as enzyme, allow at maximum one missed cleavage site, assume carbamidomethyl as a fixed modification of cysteine, and oxidized methionine and deamidation of asparagines and glutamine as variable modifications. Mass tolerance was set to 1.1 and 0.1 Da for MS and MS/MS, respectively. Data from each gel slice were searched separately. For proteins detected in several gel slices, only the highest scoring value of a single analysis is given (see Supplementary Tables 1 and 2). Proteins identified by a single peptide were listed in the tables only after manual evaluation of the fragment spectrum following the criteria suggested by Chen et al. [48]: (A) The scoring value exceeded the MASCOT homology threshold. (B) All isotopically resolved peaks with intensities higher than 5% of the maximum intensity and m/z ratios larger than that of the double or triple charged parent mass must match theoretical peptide fragments. (C) Manual interpretation of the fragment spectrum resulted in a continuous stretch of at least four amino acids.

3

Results and discussion

3.1 An improved method for glycosome purification Previously published methods for glycosome purification, based on sucrose or Percoll gradient centrifugation, resulted in a considerable degree of cross-contamination of the glycosome-enriched fractions with proteins from other subcellular compartments [9, 49–54]. Before we could analyze the glycosomal proteome by nano LC-ESI-Q-TOF-MS (nanoLC-MS/MS), it was necessary to improve the glycosome purification protocol. Many published methods for the isolation of highly purified peroxisomes use iodixanol as density gradient medium (see [55] and references therein). In iodixanol gradients, a large difference in buoyant density is found between peroxisomes and other subcellular compartments. We therefore used iodixanol (Optiprep) as a gradient medium for glycosome purification. The results are shown in Fig. 1. The gradient protein-distribution profiles showed two distinct protein peaks for both BSF (Fig. 1A, upper panel) and PCF (Fig. 1B, upper panel) trypanosomes. Glycosomecontaining fractions were identified by Western blotting using an antibody directed against the glycosomal membrane protein PEX11 [30]: glycosomes were enriched in frac© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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tion numbers 5 to 9, corresponding well to the first protein peak (Fig. 1A, B). SDS-PAGE and CBB staining of the glycosome-containing peak fractions 8 of both life forms (Fig. 1C, lanes 2) revealed a significant enrichment of specific protein bands in these fractions. To assess the purity of the glycosome-enriched fractions, Western blot analysis was performed using antibodies (see legend Fig. 1) directed against specific marker proteins from mitochondria, acidocalcisomes, and lysosomes. None of these marker proteins could be detected in the glycosome-enriched fractions (Fig. 1A and B, lower panels). Instead, they were found in fractions within the second peak of the protein distribution profile, suggesting the presence of the corresponding subcellular compartments in this particular part of the gradient. In addition, a minor portion of the glycosomes seemed to migrate in these particular fractions, as shown by the presence of PEX11. We concluded that the glycosome-enriched fractions, purified by iodixanol gradient centrifugation, were suitable for nanoLC-MS/MS analysis.

3.2 Overview of MS results In total, 10 506 and 8239 MS/MS spectra were obtained for PCF and BSF glycosomes, respectively. A first analysis of these spectra was performed by MASCOT. Identified proteins with a known or predicted function are listed in Table 1, whereas proteins without a known function (“hypothetical” proteins in the T. b. brucei genome database) are listed in Table 2. Corresponding score values, peptide matches, peptide coverage and protein masses are listed in the Supplementary Tables 1 and 2. The nanoLC-MS/MS data allowed the identification of 106 proteins in PCF glycosomes and 109 proteins in BSF glycosomes. To reduce the false positive rate to a minimum, candidates identified by a single peptide are taken as true only if a manual interpretation of the fragment spectrum matches the criteria outlined in Section 2. In total, we found 159 different proteins: 94 proteins have a known or a predicted function based on sequence similarity. Of these proteins, 39 contain a PTS1 sequence, and are therefore most likely glycosomal, whereas 12 were already known to be glycosomal even if lacking an identifiable PTS sequence (Table 1). The remaining 65 proteins had no assigned function (“hypothetical” proteins: Table 2). Sequence analysis of these proteins, using different programs for the prediction of organellar sorting signals (PSORT, PTS1, PREDATOR: available at http://www.expasy.ch) resulted in the identification of a PTS1 sequence in 10 proteins, suggesting a glycosomal localization. BLASTP analysis and comparison of the 65 proteins without known function against the PFAM (PROSITE) database revealed further the presence of conserved sequence motifs in 3 of the PTS1-containing proteins: Tb927.4.1360 showed a high similarity to different www.proteomics-journal.com

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Table 1. Proteins with known function identified by nanoLC-MS/MS. The identified proteins are grouped according to the metabolic pathways they belong to (A–P), their putative subcellular localization (Q, R, U), their function (T) or are miscellaneous (S). GeneDB accession numbers, protein descriptions and enzyme KEGG numbers are indicated. Predicted PTS1 sequences are shown between brackets. Abbreviations used are: PCF, procyclic culture form; BSF, bloodstream form; TS, targeting signal; PTS1, peroxisomal targeting signal type 1; MITO, mitochondrial targeting signal; TM, transmembrane domain (numbers of putative TM are indicated). Symbols in the PCF and BSF columns: –, not detected by nanoLC-MS/MS; 1, detected by nanoLC-MS/MS. Symbols in other columns: –, not present

Accession Number

Description

KEGG number

PCF

BSF

TS

TM

HK1, hexokinase1 HK2, hexokinase2 PGI, glucose-6-phosphate isomerase PFK, ATP-dependent phosphofructokinase ALD, fructose-bisphosphate aldolase TIM, triosephosphate isomerase GLK1, gk, glycerol kinase

2.7.1.1 2.7.1.1 5.3.1.9 2.7.1.11 4.1.2.13 5.3.1.1 2.7.1.30

1 1 1 1 1 1 1

1 1 1 1 1 1 1

2 2 PTS1 (SHL) PTS1 (AKL) 2 2 PTS1 (AKL)

2 2 2 2 2 2 2

GAPDH, glyceraldehyde 3-phosphate dehydrogenase Glycerol-3-phosphate dehydrogenase [NAD1] PGKA, P56, phosphoglycerate kinase PGKB, cPGK-8, phosphoglycerate kinase PGKC, gPGK, phosphoglycerate kinase

1.2.1.12 1.1.1.8 2.7.2.3 2.7.2.3 2.7.2.3

1 1 1 2 2

1 1 2 1 1

PTS1 (AKL) PTS1 (SKM) 2 2 PTS1 (SSL)

2 2 2 2 2

2.7.1.40 2.7.9.1 4.1.1.49

2 1 1

1 2 1

2 PTS1 (AKL) PTS1 (SRL)

2 2 2

1.1.1.37 1.3.1.6 2.3.1.61

1 1 2

2 2 1

PTS1 (SKL) PTS1 (SKI) 2

2 2 2

Ribokinase TK, transketolase Deoxyribose-phosphate aldolase G6PD, glucose-6-phosphate 1-dehydrogenase FBPase, fructose-1,6-bisphosphatase Sedoheptulose-1,7-bisphosphatase

2.7.1.15 2.2.1.1 4.1.2.4 1.1.1.49 3.1.3.11 3.1.3.37

1 1 1 1 1 1

1 2 2 2 1 2

PTS1 (CKI) PTS1 (SHL) PTS1 (SKY) 2 PTS1 (SKL) PTS1 (SKL)

2 2 2 2 2 2

Hypoxanthine-guanine phosphoribosyltransferase HGPRT, hypoxanthine-guanine phosphoribosyltransferase Guanylate kinase Inosine-5’-monophosphate dehydrogenase Inosine-5’-monophosphate dehydrogenase Adenine phosphoribosyltransferase Adenylate kinase Adenylate kinase Adenylate kinase

2.4.2.8 2.4.2.8 2.7.4.8 1.1.1.205 1.1.1.205 2.4.2.7 2.7.4.3 2.7.4.3 2.7.4.3

1 1 1 1 1 1 1 1 2

1 2 2 2 1 2 2 1 1

PTS1 (SKL) PTS1 (AKL) PTS1 (SKL) PTS1 (AKL) PTS1 (SKL) PTS1 (SRL) PTS1 (SKL) 2 2

2 2 2 2 2 2 2 2 2

2.4.2.10 4.1.1.23

1

1

PTS1 (SKL)

2

(A) Glycolysis Tb10.70.5820 Tb10.70.5800 Tb927.1.3830 Tb927.3.3270 Tb10.70.1370 Tb11.02.3210 Tb09.211.3540/ 3550/3560/ 3570/3580 Tb927.6.4300 Tb927.8.3530 Tb927.1.720 Tb927.1.710 Tb927.1.700

(B) Pyruvate Metabolism Tb10.61.2680 Tb11.02.4150 Tb927.2.4210

PYK, pyruvate kinase PPDK, pyruvate phosphate dikinase PEPCK, phosphoenolpyruvate carboxykinase

(C) Krebs Cycle Enzymes Tb10.61.0980 Tb927.5.930 Tb11.01.3550

gMDH, glycosomal malate dehydrogenase FRDg, NADH-dependent fumarate reductase 2-Oxoglutarate dehydrogenase, E2 component, dihydrolipoamide succinyltransferase

(D) Pentose Phosphate Pathway/Calvin-Benson Cycle Tb11.03.0090 Tb927.8.6170 Tb927.7.5680 Tb10.70.5200 Tb09.211.0540 Tb927.2.5800 (E) Purine Salvage Tb10.70.6660 Tb10.70.6540 Tb10.6k15.3960 Tb927.5.2080 Tb10.61.0150 Tb927.7.1790 Tb10.70.1200 Tb10.70.7330 Tb927.2.5660

(F) Pyrimidine Metabolism Tb927.5.3810

Orotidine-5-phosphate decarboxylase/orotate phosphoribosyltransferase

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Table 1. Continued

Accession Number

Description

KEGG number

PCF

BSF

TS

TM

5.3.1.8

1

2

PTS1 (SHM)

2

2.5.1.26

1

1

PTS1 (AHL)

2

3.1.1.5

1

1

PTS1 (SKS)

1

2 2 2 2 1.1.1.42

1 1 1 1 1

2 2 2 2 1

PTS1 (ANM) 2 PTS1 (TKM) PTS1 (ARL) PTS1 (SKV)

2 2 2 2 2

1.15.1.1

1

2

2

2

1.1.1.103

2

1

PTS1 (PSL)

2

2.7.1.36

1

2

PTS1 (SKL)

2

2.7.1.36 2.7.3.3

2 2

1 1

PTS1 (SNL) 2

2 2

2 1.1.1.146

1 1

1 2

2 2

4 2

2 2 2 2 2 2 2 2 2 2

1 2 1 1 1 2 1 1 1 1

1 1 2 1 1 1 1 2 2 2

2 2 2 2 2 2 2 2 2 2

1 3 2 2 2 3 2 1 4 3

Hydrolase, alpha/beta fold family ACBP, acyl-CoA binding protein Acetyltransferase NUDIX hydrolase Heat shock protein 70

2 2 2 2 2

1 1 2 1 2

2 2 1 2 1

PTS1 (AKL) PTS1 (SNL) PTS1 (SKY) PTS1 (SSI) PTS1 (SSL)

2 2 2 2 2

60S acidic ribosomal subunit protein Chaperone protein DNAJ

2 2

2 2

1 1

2 2

2 2

(G) Mannose and Fructose Metabolism Tb11.01.6410

Phosphomannose isomerase

(H) Glycerolipid Metabolism Tb927.6.1500

DHAP, alkyl-dihydroxyacetone phosphate synthase

(I) Phospholipid Degradation Tb927.8.6390

LysoPLA, lysophospholipase

(J) Trypanothion Metabolism Tb927.7.7500 Tb927.3.3780 Tb927.5.300 Tb927.7.1140 Tb11.03.0230

Iron/ascorbate oxidoreductase family protein Tryparedoxin Iron/ascorbate oxidoreductase family protein GPXIII, trypanothione/tryparedoxin dependent peroxidaseIII IDH, isocitrate dehydrogenase

(K) Superoxide Metabolism Tb11.01.7550

Iron superoxide dismutase

(L) Threonine Metabolism Tb927.6.2790

L-Threonine 3-dehydrogenase

(M) Mevalonate Pathway Tb927.4.4070

Mevalonate kinase

(O) Arginine Metabolism Tb09.160.4570 Tb09.160.4560

AK, arginine kinase AK, arginine kinase

(P) Steroid Metabolism Tb927.3.1840 Tb11.01.1780

3-Oxo-5-alpha-steroid 4-dehydrogenase 11-beta-hydroxy-steroid dehydrogenase

(Q) Membrane Proteins Tb09.211.2730 Tb11.03.0030 Tb927.3.2340 Tb10.100.0130 Tb11.01.3370 Tb09.160.0620 Tb10.61.0440 Tb09.211.1750 Tb10.389.0690 Tb10.61.1810/ 1820/1830

GIM5A ABCD3, ABC transporter, PMP70 PEX2 PEX14 PEX11 Peroxisomal membrane protein 4 PEX12 Mitochondrial carrier protein, Pi Carrier Mitochondrial carrier protein, Dicarboxylate Carrier Mitochondrial carrier protein, ATP/ADP Carrier

(R) Miscellaneous/PTS1 Tb927.5.2370 Tb927.4.2010 Tb927.1.4490 Tb927.5.4350 Tb11.01.3110 (S) Miscellaneous Tb11.46.0001 Tb11.01.6780

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Table 1. Continued

Accession Number

Description

KEGG number

PCF

BSF

TS

TM

Tb10.70.5650 Tb11.03.0410 Tb10.100.0090 Tb11.02.5450

TEF1, elongation factor 1-alpha eIF-5A, eukaryotic translation initiation factor 5a Vacuolar ATP synthase Glucose-regulated protein 78

2 2 2 2

1 2 2 2

1 1 1 1

2 2 2 2

2 2 2 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

1 1 1 2 2 1 2 1 1 1 1 1 1 1 1

1 1 1 1 1 2 1 2 1 2 2 1 2 1 1

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 1.1.1.8

1 1 1 2 2 1 1 2

1 2 2 1 1 2 1 1

MITO MITO MITO MITO 2 MITO 2 MITO

2 2 2 1 2 2 2 2

(T) Structural Proteins Tb927.3.4290 Tb927.8.5010 Tb11.01.5100 Tb927.5.4480 Tb927.8.4640 Tb927.5.4170 Tb10.406.0460 Tb10.406.0330 Tb11.47.0034 Tb11.50.0007 Tb09.211.4511 Tb11.01.4621 Tb09.160.4520 Tb927.1.2330 Tb927.1.2340

PFR1, PFRC, 73 kDa paraflagellar rod protein PFR2, PFRA, 69 kDa paraflagellar rod protein Paraflagellar rod component Paraflagellar rod component Par4 Flagellar protofilament ribbon protein Histone H4 Histone H2B Histone H2B Radial spoke protein 3 Dynein light chain Kinetoplastid membrane protein KMP-11 Calmodulin Calmodulin beta Tubulin alpha Tubulin

(U) Mitochondrial Proteins Tb927.1.2670 Tb927.7.7430 Tb927.3.1380 Tb10.6k15.3640 Tb927.6.3800 Tb927.8.7170 Tb10.70.0280 Tb11.02.5280

Axoneme central apparatus protein ATP Synthase alpha chain, mitochondrial precursor ATP Synthase beta chain, mitochondrial precursor AOX, TAO, alternative oxidase Heat shock 70 kDa protein, mitochondrial precursor Inositol polyphosphate 1-phosphatase HSP60, chaperonin Hsp60, mitochondrial precursor Glycerol-3-phosphate dehydrogenase

aldose-1-epimerase proteins, whereas Tb09.160.4480 and Tb09.160.4460 were found to be similar to nuclear LIM-factor-interactor interacting-proteins (Table 2). Of all characterized peptides, 16% could be assigned to known non-glycosomal proteins (Supplementary Tables 1 and 2). These contaminants include abundant proteins like chaperones, transcription factors, cytoskeletal and flagellar proteins, and a few mitochondrial membrane proteins (Table 1).

3.3 Glycolysis: Going from glucose to glycerate 3-phosphate Hallmark of the glycosome is the compartmentalization of the first seven to nine enzymes of the glycolytic pathway [11, 16, 50, 56]. Depending on the life form, glucose is metabolized in the glycosome either to glycerate 1,3bisphosphate or to glycerate 3-phosphate, respectively (see Fig. 2A). The further conversion of these metabolic intermediates to pyruvate was postulated to be cytosolic in both © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

life forms [11, 33]. All glycolytic enzymes, previously reported to be glycosomal, were identified by nanoLC-MS/ MS (Table 1A, Fig. 2A). The first enzyme of the glycolytic pathway, hexokinase (HK, EC 2.7.1.1), catalyzes the transfer of the g-phosphoryl group of ATP to glucose. The glucose 6-phosphate formed is metabolized either by the glycolytic pathway (Fig. 2A) or enters the oxidative branch of the pentose phosphate pathway (Fig. 2D). Interestingly, our analyses revealed the presence of two distinct hexokinases in the glycosomal fractions of both PCF and BSF trypanosomes: HK1 and HK2, encoded by the tandemly-arranged genes Tb10.70.5820 and Tb10.70.5800, respectively. These proteins share 97.7% amino acid identity and were distinguished by MS/MS spectra of three peptides unique for HK1 and two peptides unique for HK2. Why the glycosomes of both life forms contain two different hexokinases is not clear at this point. The last step of the glycolytic pathway, e.g. the conversion of glycerate 1,3-bisphosphate to glycerate 3-phosphate (Fig. 2A), is catalyzed by phosphoglycerate kinase (PGK, EC 2.7.2.3). The genome of T. b. brucei contains three different www.proteomics-journal.com

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Table 2. Proteins without known function (“hypothetical” proteins) identified by nanoLC-MS/MS. GeneDB accession numbers, BLASTP hits, and motifs predicted by PFAM are indicated. Predicted PTS1 sequences are shown between brackets. Abbreviations used are: PCF, procyclic culture form; BSF, bloodstream form; TS, targeting signal; PTS1, peroxisomal targeting signal type 1; TM, transmembrane domain (numbers of putative TM are indicated); Exp, Expectation. Symbols in the PCF and BSF columns: –, not detected by nanoLC-MS/MS; 1, detected by nanoLC-MS/MS. Symbols in other columns: –, not present

Accession Number

PCF

BSF

TS

TM

BLASTP

Motifs

Tb09.160.4460

1

1

0

Tb09.160.4480

1

–

Tb09.211.0170 Tb09.211.1470 Tb09.211.2250 Tb10.26.0680 Tb10.61.1260 Tb10.61.1550 Tb10.61.2210 Tb10.61.2220 Tb10.6k15.0400

– 1 – 1 – – 1 1 1

1 1 1 1 1 1 1 1 –

0 0 0 0 0 0 0 0 0

Nuclear LIM factor interactor-interacting protein (Phytophthora sojae) Exp 8e-15 Nuclear LIM factor interactor-interacting protein (Phytophthora sojae) Exp 8e-15 – – – – – – – – –

NLI interacting factor-like phosphatase Exp 3.1e-09 NLI interacting factor-like phosphatase Exp 9e-10 – – – – – – – – –

Tb10.6k15.0810 Tb10.6k15.1510 Tb10.6k15.2920

1 1 –

1 – 1

PTS1 (SRL) PTS1 (SRL) – – – – – – – – PTS1 (ARL) – – –

0 0 0

– – –

Tb10.70.1080 Tb10.70.5560

– 1

1 1

– –

0 0

– –

Tb10.v4.0053

1

1

–

0

Tb10.v4.0248 Tb11.01.0840 Tb11.01.1210 Tb11.01.1625 Tb11.01.2800 Tb11.01.3000 Tb11.01.4030 Tb11.01.6740 Tb11.01.7750 Tb11.02.0140 Tb11.02.1260 Tb11.02.2490 Tb11.02.2530 Tb11.02.4320 Tb11.02.4380 Tb11.02.5460 Tb11.02.5550

– 1 – 1 1 – 1 1 – – – – 1 – – – 1

1 1 1 – 1 1 1 1 1 1 1 1 – 1 1 1 –

– – – – – – – – – – – – – – – – –

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Microtubule-associated protein p320 (Trypanosoma brucei) Exp 5e-168 – – – – – – – – – – – – – – – – –

– – Repeat of unknown function (DUF1126) Exp 1.8e-11 – Protein of unknown function (DUF667) Exp 1.1e-146 –

Tb11.03.0470 Tb11.1220 Tb11.46.0011 Tb11.47.0006 Tb927.1.5000 Tb927.2.2160 Tb927.2.2770 Tb927.3.2310 Tb927.3.3770

– – – 1 1 – – 1 –

1 1 1 1 – 1 1 1 1

– – – – – – – – PTS1 (SRY)

0 0 0 0 0 0 0 0 0

– – – – – – – – –

0

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

– – – – – – – – – – Dpy-30 motif Exp 9.7e-10 – – – – – WD domain, G-beta repeat Exp 5.2e-07 – – – – – – – – –

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Table 2. Continued

Accession Number

PCF

BSF

TS

TM

BLASTP

Motifs

Tb927.3.3790

1

1

0

–

–

Tb927.3.4420

1

1

0

–

–

Tb927.4.1360

1

–

0

Tb927.4.1740 Tb927.4.2840 Tb927.4.4040 Tb927.4.4690 Tb927.4.4700 Tb927.5.1230 Tb927.5.2650

– 1 – 1 1 – 1

1 – 1 1 1 1 –

0 0 0 0 0 0 0

Aldose 1-epimerase-like (Oryza sativa) Exp 3e-38 – – – – – – –

Aldose 1-epimerase Exp 3.4e-18 – – – – – – –

Tb927.5.2850 Tb927.5.2950

– –

1 1

PTS1 (SRY) PTS1 (SKM) PTS1 (SKM) – – – – – – PTS1 (SHL) – –

0 0

– –

Tb927.6.2200

1

–

0

–

– Repeat of unknown function (DUF1126) Exp 2.5e-14 DJ-1/PfpI family Exp 3e-23

Tb927.6.4140 Tb927.6.4520 Tb927.7.2190 Tb927.7.3740 Tb927.8.1550 Tb927.8.4580 Tb927.8.6240 Tb927.8.6640

1 1 – – 1 1 1 1

1 – 1 1 1 1 – –

0 0 2 0 0 0 0 0

– – – – – – – –

– – – – – – – NAD binding 4 Exp 2.3e-87

Tb927.8.6660

1

1

0

–

–

PTS1 (SKF) – – – – – – – PTS1 (SSL) –

PGK genes [57]. The first gene, PGKA, encodes a minor glycosomal variant (PGKA) that is expressed at low levels in both BSF and PCF T. b. brucei [57–59]. The second gene, PGKB, encodes the major cytosolic enzyme (PGKB), which is present only in PCF T. b. brucei [57, 60]. The third gene, PGKC, encodes the major glycosomal enzyme (PGKC), and is expressed only in BSF T. b. brucei [35, 61]. The amino acid sequences of PGKB and PGKC are rather similar (89.1% amino acid identity). Our results showed that, in agreement with previous reports, PCF glycosomes contained PGKA, whereas PGKC was found exclusively in BSF glycosomes (Table 1A). However, in contrast to previous reports, not PGKA, but PGKB was detected in BSF glycosomes (Table 1A). The identification of PGKB was based on the presence of two almost identical peptides, showing only one amino acid substitution: T414 in PGKB opposed to A414 in PGKC. Since the amino acid substitution is caused by a single nucleotide change, and all published experimental evidence suggests that PGKB is cytosolic, it is likely that we have in fact simply detected a difference between the two alleles of PGKC in the diploid T. b. brucei. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

NanoLC-MS/MS revealed further the presence of an aldose-1-epimerase, encoded by Tb927.4.1360 and bearing a PTS1 sequence, in PCF glycosomes (Table 2). Common hexose sugars such as glucose exist in two forms in aqueous solution, the so-called a- and b-anomers. Aldose-1-epimerase or mutarotase (EC 5.1.3.3) catalyzes the interconversion of these anomers and is a key enzyme of carbohydrate metabolism since many enzymes exhibit specificity for one anomer or the other [62, 63]. Aldose-1-epimerase supports energy generation in organisms whose metabolic enzymes are biased towards a specific aldose anomer. For example, in erythrocytes the b-anomer of glucose is more rapidly metabolized by glycolysis as the a-anomer [64–66]. Expression studies in the fungus Rhizopus nigricans showed that aldose-1-epimerase is repressed by high glucose concentrations and is de-repressed during deficiency of glucose [67]. Detection of this enzyme in PCF, but not in BSF T. b. brucei, suggests a similar type of regulation: BSF trypanosomes grow in high glucose (3–5 mM in blood [68, 69], 10 mM in the culture medium), and lack accordingly the aldose-1-epimerase, whereas the medium for the aldose-1-epimerase expressing PCF trypanosomes contains 30 times less glucose (0.1–0.3 mM). www.proteomics-journal.com

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Figure 2. Overview of the BSF and PCF glycosomal proteomes of T. b. brucei. Enzymatic steps found only in the PCF glycosome are indicated by blue arrows, those found only in the BSF glycosome by red arrows, and those occurring in glycosomes of both life forms are indicated by black arrows. Green arrows indicate enzymes that were not detected by nanoLC-MS/MS. The different pathways are indicated by black capital characters and are boxed grey: see corresponding sections in Table 1. KEGG numbers (see also Table 1) of the involved enzymes are indicated alongside the arrows, and are red if the protein contains a PTS1 sequence, and black if not. For phosphoglycerate kinase (EC 2.7.2.3): A, B and C (red: contains PTS1) indicate the different isoforms. Membrane proteins are indicated by blue bullets if found only in PCF cells, red bullets if they are present in BSF cells, and black bullets if they are present in both life forms. Putative transporters are indicated by black bullets with white question marks: the transport-direction is indicated by dashed grey arrows. Abbreviations used are: AOX, alternative oxidase; ASC, ascorbate; MDA, monodehydroxyascorbate; AOR, ascorbate oxidoreductase; 2KG, 2-ketoglutarate; 1-acylGPC, 1-acyl-glycero-phosphocholine; PRPP, 5-phosphoribosyl 1-pyrophosphate; ox, oxidized; red, reduced.

3.4 The fate of glycerone 3-phosphate Within the glycosome, glycerone 3-phospate (DHAP) is converted to glycerol by the sequential action of nicotinamide adenine dinucleotide (NAD)-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) and glycerol kinase (EC 2.7.1.30) (Fig. 2A). Glycerol-3-phosphate dehydrogenase catalyses the interconversion of DHAP and glycerol 3-phosphate (Fig. 2A). In BSF glycosomes, this enzyme plays an important role in the re-oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) formed by the glyceraldehyde-3-phosphate dehydrogenase reaction, and is involved in the glycerol-3-phosphate/DHAP shuttle, which was reported previously to be essential for survival of the © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

parasite [16, 70]. In addition, PCF glycosomes were suggested to contain a functional glycerol-3-phosphate/DHAP shuttle [16]. NanoLC-MS/MS confirmed the presence of NAD-dependent glycerol-3-phosphate dehydrogenase (Tb927.8.3530) in the glycosomes of both life forms (Table 1A). Glycerol kinase catalyzes the interconversion of glycerol 3-phosphate to glycerol and was reported previously to be localized in BSF and PCF glycosomes [12, 52, 71]. NanoLCMS/MS confirmed the presence of glycerol kinase in the glycosomes of both life forms (Table 1A). The T. b. brucei genome contains five almost identical tandemly-arranged glycerol kinase-encoding genes (Tb09.211.3540, Tb09.211.3550, Tb09.211.3560, Tb09.211.3570, and Tb09.211.3580), each containing the same PTS1 (AKL) sequence. However, due to www.proteomics-journal.com

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the high sequence similarity (99.8% amino acid identity) we were unable to discriminate between the five different glycerol kinases by nanoLC-MS/MS. The detection of both glycerol-3-phosphate dehydrogenase and glycerol kinase in PCF glycosomes is consistent with the previously reported observation that PCF trypanosomes can metabolize glycerol [20, 72]. 3.5 Pyruvate metabolism In both T. b. brucei life forms, glycerate 3-phosphate is converted in the cytosol to phosphoenolpyruvate (PEP) by glycerate-3-phosphate mutase (EC 5.4.2.1) and enolase (EC 4.2.1.11) (Fig. 2A). In BSF trypanosomes, a cytosolic pyruvate kinase (EC 2.7.1.40) converts PEP to pyruvate, which is secreted to the medium as a metabolic endproduct [73]. In PCF trypanosomes, however, PEP is metabolized via two different pathways (Fig. 2B). In the first pathway, a cytosolic pyruvate kinase converts PEP to pyruvate, which is channeled to the mitochondria where it is decarboxylated to acetate [23, 73]. In the second pathway, PEP is re-imported into the glycosome where it is converted to oxaloacetate by phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49). Oxaloacetate is metabolized further in the glycosome to succinate, which is finally secreted to the medium (Fig. 2C) [74]. Remarkably, nanoLC-MS/MS analysis revealed the presence of both phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase in the glycosomes of BSF trypanosomes (Table 1B, Fig. 2B). PEPCK contains a PTS1 sequence (SRL), but so far has been reported to be present only in PCF glycosomes [50, 75], whereas pyruvate kinase lacks a peroxisomal targeting signal and was previously reported to be exclusively cytosolic in both life forms [33]. The unexpected presence of both PEPCK and pyruvate kinase in BSF glycosomes suggests two possible biochemical scenarios (Fig. 2B). In the first scenario, PEP re-enters the glycosome were it is converted to either oxaloacetate by PEPCK or to pyruvate by pyruvate kinase. In either way, an additional ATP would be generated in the glycosome. In the second scenario, not PEP but pyruvate re-enters the glycosome, which is converted to oxaloacetate by the combined action of both pyruvate kinase and PEPCK. This, however, would result in loss of glycosomal ATP, suggesting that the first scenario is energetically more favorable. PCF trypanosomes were reported previously to contain a glycosomal pyrophosphate-dependent pyruvate phosphate dikinase (PPDK, EC 2.7.9.1, Fig. 2B), which catalyzes the reversible conversion of pyruvate to PEP [76]. It has been postulated that in T. b. brucei the equilibrium of this reaction is predominantly towards pyruvate formation: the rationale for this assumption was mainly the need for removal of pyrophosphate (PPi) formed during glycosomal b-oxidation of fatty acids in trypanosomes [76, 77]. However, for both BSFand PCF T. b. brucei, we could not detect any of the four key-enzymes involved in fatty acid b-oxidation, suggesting that this pathway is absent in the glycosomes of this parasite (see Section 3.18). © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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An alternative function of PPDK could be the detoxification of PPi formed during purine salvage and pyrimidine biosynthesis. Enzymes involved in both pathways were detected by nanoLC-MS/MS (see Sections 3.8 and 3.9) in the glycosome of both life forms, and were reported previously to be inhibited by PPi [78]. However, BSF glycosomes lack PPDK, and depletion of this enzyme in PCF T. b. brucei was shown not to be lethal [19], suggesting that this enzyme is not essential for pyrophosphate-detoxification at all. 3.6 Maintenance of the glycosomal NADH balance In vivo labeling-experiments, using 13C-labeled glucose as substrate, suggested that in glycosomes of PCF trypanosomes oxaloacetate is metabolized to succinate (Fig. 2C), and that this pathway is involved in the maintenance of the glycosomal NADH balance [74, 79]. Of the three Krebs cycle enzymes needed for this conversion, e.g. malate dehydrogenase (EC 1.1.1.37), fumarase (EC 4.2.1.2), and NADHdependent fumarate reductase (EC 1.3.1.6), only malate dehydrogenase (gMDH) and fumarate reductase (FRDg) were shown experimentally to be localized in the glycosomes [52, 79]. NanoLC-MS/MS analysis confirmed the presence of both gMDH and FRDg in the glycosomes of PCF trypanosomes (Table 1C), whereas fumarase could not be detected. The genome of T. b. brucei contains two fumarase-encoding genes: Tb927.3.4500 and Tb11.02.2700. The deduced amino acid sequences lack peroxisomal targeting signals, indicating the absence of these proteins in the glycosome. Coustou et al. [74] showed recently by knockout of the glycosomal FRDgencoding gene that succinate, derived from glucose, is formed in the mitochondrion from glycosomal malate, implying the exchange of malate between the two organelles. The lack of a glycosomal fumarase confirms that malate has to be transferred to the mitochondrion, were it is converted to fumarate by a mitochondrial fumarase, and finally to succinate by the mitochondrial fumarate reductase. Knockout of the mitochondrial fumarate reductase revealed also that part of the formed succinate is synthesized in the glycosome [74, 79] This, together with the lack of glycosomal fumarase, suggests that fumarate has to be transferred back to the glycosome were it is converted to succinate by the glycosomal FRDg. In this way, the glycosomal NADH balance would be preserved, even in the absence of fumarase in this organelle. 3.7 Pentose phosphate pathway and Calvin-Benson cycle The pentose phosphate pathway (PPP) provides the cell with reductive equivalents [the reduced form of NADP (NADPH)], nucleic acid precursors such as ribose 5-phosphate and 5-phosphoribosyl 1-pyrophosphate (PRPP), and several metabolic intermediates such as fructose 6-phosphate and glyceraldehyde 3-phosphate. www.proteomics-journal.com

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For PCF T. b. brucei, the first three enzymes of the oxidative branch of the pentose phosphate pathway have been characterized [80, 81]. Heise et al. [80] showed that 50% of the glucose-6-phosphate 1-dehydrogenase (G6PDH, EC 1.1.1.49) activity, the first enzyme of the oxidative PPP branch, was found in the glycosomes, whereas the rest of the activity was cytosolic. Of the second enzyme, the 6-phosphogluconolactonase (EC 3.1.1.31), only 15% of its activity could be detected in the glycosome, whereas the remaining 85% was found to be cytosolic [81]. The third enzyme of the oxidative PPP branch, 6-phosphogluconate dehydrogenase, (EC 1.1.1.44) was found exclusively in the cytosol [80]. NanoLC-MS/MS analysis confirmed the presence of glucose-6-phosphate 1-dehydrogenase (Tb10.70.5200) and the absence of 6-phosphogluconate dehydrogenase in PCF glycosomes (Table 1D). The 6-phosphogluconolactonase, however, could not be detected. Considering the high sensitivity of the nanoLC-MS/MS method used, we assume that the reported low (15%) glycosomal phosphogluconolactonase activity is due to cross-contamination with cytosolic enzyme [81]. For BSF glycosomes, no enzymes of the oxidative PPP branch could be detected, which is in agreement with the reported cytosolic localization of these enzymes in this life form [82]. The first enzyme of the non-oxidative (reductive) PPP branch, transketolase (EC 2.2.1.1), catalyzes the reversible transfer of a two-carbon unit from D-xylulose-5-phosphate to either D-ribose-5-phosphate or D-erythrose-4-phosphate, generating glyceraldehyde 3-phosphate and sedoheptulose 7phosphate or fructose 6-phosphate (Fig. 2D). Cronin et al. [82] reported that in T. b. brucei transketolase activity is only present in the PCF trypanosome. In the related kinetoplastid Leishmania, transketolase activity was found in both the glycosome and the cytosol [83]. In agreement with these reports, we detected transketolase in the PCF, but not in the BSF glycosomes. The second enzyme of the non-oxidative PPP branch is transaldolase (EC 2.2.1.2), which converts sedoheptulose 7phosphate and glyceraldehyde 3-phosphate to fructose 6phosphate and erythrose 4-phosphate, respectively. Transaldolase activity has been reported previously for BSF and PCF T. b. brucei, but its subcellular localization was not determined [82]. NanoLC-MS/MS could not detect this protein in the glycosomes of both life forms, suggesting that glycosomes lack the non-oxidative PPP branch. Hannaert et al. [84] reported the presence of sedoheptulose-1,7-biphosphatase (EC 3.1.3.37) and fructose-1,6bisphosphatase (EC 3.1.3.11) in PCF trypanosomes. These enzymes are involved in the Calvin-Benson cycle, and were until now only found in photosynthetic organisms. Both proteins contain a PTS1 sequence, and nanoLC-MS/MS analysis (Table 1D) demonstrated their glycosomal localization. Together with transketolase, these enzymes constitute part of the Calvin-Benson cycle (Fig. 1D), which regenerates ribulose 1,5-bisphosphate from triose-phosphates [85, 86]. In PCF glycosomes, however, the cycle stops with the formation © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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of ribose-5-phosphate (Fig. 1D): other enzymes like ribose-5phosphate isomerase (EC 5.3.1.6) and ribose-phosphate diphosphokinase (EC 2.7.6.1), which convert ribose-5-phosphate to ribulose-5 phosphate and 5-phosphoribosyl 1-pyrophosphate (PRPP), respectively, could not be detected by nanoLC-MS/MS. The corresponding genes, Tb11.01.0700 and Tb927.5.3170, were found in the T. b. brucei genome, but the deduced amino acid sequences lack a PTS1 sequence, indicating that these proteins are not targeted to the glycosome. Additionally, nanoLC-MS/MS analysis revealed the presence of a deoxyribose-phosphate aldolase (EC 4.1.2.4) in PCF glycosomes, and the presence of a ribokinase (EC 2.7.1.15) in glycosomes of both life forms (Table 1D). Deoxyribose-phosphate aldolase catalyzes the conversion of glyceraldehyde 3phosphate to 2-deoxy-D-ribose-5-phosphate, whereas ribokinase catalyses the conversion of D-ribose 5-phosphate to Dribose (Fig. 1D). D-ribose 5-phosphate and 2-deoxy-D-ribose 5-phosphate are precursors for the biosynthesis of nucleic acids. 3.8 Purine salvage T. b. brucei depends entirely on the salvage of purine bases and nucleosides provided by the different hosts [78]. Enzymes involved in the purine salvage pathway are hypoxanthine-guanine/adenine phosphoribosyl transferases, adenine and adenosine deaminases, purine nucleoside kinases and nucleoside hydrolases [87, 88]. Hypoxanthine-guanine phosphoribosyl transferases (EC 2.4.2.8) catalyze the single-step conversion of nucleotides (XMP, IMP, GMP and AMP) to their nucleobases (xanthine, hypoxanthine, guanine and adenine). A similar reaction is catalyzed by adenine phosphoribosyl transferases (EC 2.4.2.7), which convert AMP to adenine and GMP to guanine (see Fig. 1E). Phosphoribosyl transferase activity was shown previously to be associated with the glycosomes of trypanosomes [88]. Our results revealed that both adenine- and hypoxanthine-guanine phosphoribosyl transferases are present in PCF glycosomes, whereas in BSF glycosomes only the hypoxanthine-guanine-specific enzyme was detected (Table 1E). IMP-dehydrogenase, AMP-deaminase and GMP-synthetase are involved in purine nucleotide interconversion [78]. IMP-dehydrogenases (EC 1.1.1.205) interconvert IMP to XMP. The nanoLC-MS/MS results indicated the glycosomal presence of two different IMP-dehydrogenases (Tb927.5.2080 and Tb10.61.0150), containing each a PTS1 sequence (Table 1E). These proteins share only 31% amino acid identity and can easily be distinguished by nanoLC-MS/ MS. The first IMP-dehydrogenase was detected exclusively in PCF glycosomes, whereas the second was found in the glycosomes of both life forms. AMP-deaminase and GMP-synthetase, which interconvert AMP to IMP and XMP to GMP, were not found in the glycosomes, although the corresponding genes are preswww.proteomics-journal.com

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ent in the T. b. brucei genome [47]. In addition, nucleoside kinase and nucleoside phosphorylase, involved in the twostep conversion of nucleotides to nucleobases via nucleosides, could not be detected in the glycosomes of both life forms, although also in this case the corresponding genes are present in the genome. The presence of phosphoribosyl transferases and IMPdehydrogenases in the glycosomes of both T. b. brucei life forms confirms that this organelle is involved in purine salvage. 3.9 Pyrimidine metabolism The last two steps of pyrimidine biosynthesis, the conversion of orotate to UMP, are catalyzed by an orotate-phosphoribosyl transferase (EC 2.4.2.10) and an orotidine-5’-phosphate decarboxylase (EC4.1.1.23) (Fig. 1F). For T. b. brucei, both enzyme activities were found to be associated with the glycosome [89]. Analysis of the T. b. brucei genome showed, that orotate phosphoribosyltransferase and orotidine-5’phosphate decarboxylase are not encoded by two different genes, but instead are encoded by a single gene (Tb927.5.3810). NanoLC-MS/MS analysis confirmed the glycosomal localization of the encoded bifunctional protein, which contains a PTS1 sequence, in both BSF and PCF trypanosomes (Table 1F). The glycosomal localization of the two last steps of the pyrimidine metabolism implies the transport of orotate and PRPP across the glycosomal membrane. 3.10 D-Mannose metabolism Next to glucose, T. b. brucei can also metabolize D-mannose [90]. The two enzymes involved are hexokinase, which in trypanosomes is exclusively compartmentalized in the glycosome (see Section 3.3), and phosphomannose isomerase (EC 5.3.1.8) (Fig. 2G). Phosphomannose isomerase contains a PTS1 sequence (Table 1G), suggesting a glycosomal localization. The product of the phosphomannose isomerase reaction, D-fructose 6-phosphate, can be metabolized further by the glycolytic pathway. Hara et al. [90] reported that in BSF trypanosomes, because of a relatively low phosphomannose isomerase activity, mannose 6-phosphate accumulated, leading to growth inhibition. Correspondingly, we were unable to detect this enzyme in BSF glycosomes, confirming the low abundance of the protein. In PCF glycosomes, however, phosphomannose isomerase could be detected (Table 1G), suggesting that this life form is capable of using mannose for energy metabolism. 3.11 Glycerol-ether lipid biosynthesis Trypanosomes are able to take up glycerol-ether lipids from their host or can synthesize them de novo [91]. The first three steps in the de novo biosynthesis pathway, the conversion of glycerone 3-phosphate (DHAP) to 1-alkyl-glycerol-3-phos© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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phate, were reported previously to be associated with the glycosome [91–93]. Of the involved enzymes, only alkyl-glycerone-phosphate synthetase (EC 2.5.1.26) was detected by nanoLC-MS/MS (Table 1H). This enzyme, encoded by Tb927.6.1500, contains a PTS1 sequence, and was found in the glycosomes of both BSF and PCF trypanosomes (Table 1H). The other two enzymes, glycerone-phosphate acyl-transferase (EC 2.3.1.42) and nicotinamide adenine dinucleotide phosphate (NADP)-dependent 1-acyl-glycerol-3phosphate oxidoreductase (EC 1.1.1.101), which catalyze the first and third step of the pathway, were not detected in the glycosomes of both life forms. The culture medium contains sufficient glycerol-ether lipids, which can be taken up by T. b. brucei. The absence of several enzymes of the glycerol-ether lipid biosynthesis pathway suggests that part of this pathway is down-regulated. Such a regulation of the glycerol-ether lipid metabolism in T. b. brucei has not been reported previously. 3.12 Phospholipid degradation BSF trypanosomes were shown to utilize exogenous l-acyllysophosphatidylcholine (lyso-PtdCho) via two routes: (1) lysophospholipase A catalyzes the de-acylation of lysoPtdCho to form free fatty acids and glycerophosphocholine, and (2) an acyl-CoA-lyso-PtdCho acyltransferase catalyzes the acyl-CoA-induced acylation of lyso-PtdCho to form phosphatidylcholine [94, 95]. These enzymes were reported previously to be associated with the outer membrane of T. b. brucei [94]. Interestingly, our nanoLC-MS/MS data indicated that lysophospholipase A (route 1) is also associated with the glycosomes of both BSF and PCF trypanosomes (Table 2). Analysis of its amino acid sequence revealed the presence of a PTS1 (SKS) sequence, confirming its glycosomal localization. Additionally, a putative transmembrane domain was predicted, suggesting that this protein is localized in the glycosomal membrane. An acyl-CoA-lyso-PtdCho acyltransferase was not detected, indicating that the second route is absent in the glycosomes. The fatty acids and glycerophosphocholine formed by the glycosomal lyso-phospholipase A could provide material for glycosomal membrane biogenesis. 3.13 Detoxification of oxygen radicals and superoxides Superoxide dismutases (SOD) are metallo-enzymes that degrade toxic reactive oxygen intermediates, generated by several oxidative enzymes and during the auto-oxidation of various biomolecules, to oxygen and H2O2 [96]. In Phytomonas, a plant trypanosome, SOD activity was found to be mainly located in the cytosol and, to a lesser extent, in the glycosomes [97]. Here, we report the presence of an iron-dependent SOD (EC 1.15.1.1) in the glycosomes of PCF trypawww.proteomics-journal.com

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nosomes (Table 1K). The peroxides (H2O2) formed during the SOD reaction can be scavenged by the trypanothione metabolism: in T. b. brucei an unique thiol metabolism is found in which the glutathione/glutathione reductase couple, found in most other organisms, is replaced by trypanothione (N1,N8-bis(glutathioneyl)spermidine) and the NADPH-dependent enzyme trypanothione reductase [98, 99]. Reducing equivalents are transferred via trypanothione and tryparedoxin to a trypanothione/tryparedoxin-dependent peroxidase-III (Fig. 2J), which is concomitantly reduced and converts hydroperoxides to molecular oxygen and H2O [98, 100, 101]. Both tryparedoxin (Tb927.3.3780) and trypanothione/ tryparedoxin-dependent peroxidase-III (Tb927.7.1140) were detected in PCF glycosomes (Table 1J). The trypanothione/ tryparedoxin-dependent peroxidase-III was previously reported to be exclusively present in the mitochondria of PCF trypanosomes [101]. However, closer inspection of the published Western blot (Fig. 3B in [101]) revealed that part of the peroxidase-III protein was also present in the glycosome. Peroxidase-III contains both a cleavable N-terminal mitochondrial targeting signal and a conserved PTS1 sequence (ARL), which supports a dual subcellular localization of this protein in T. b. brucei [101]. Trypanothione reductase, the first enzyme of the trypanothione metabolism, could not be detected in the glycosomes of both life forms. The lack of this enzyme in the glycosomes suggests that not oxidized trypanothione, but reduced trypanothione enters the glycosome of PCF trypanosomes, implying the presence of a glycosomal trypanothione transporter (see Section 3.17). The T. b. brucei genome contains 8 different iron-ascorbate-oxidoreductase-encoding genes: Tb927.2.6180, Tb927.2.6210, Tb927.2.6230, Tb927.2.6270, Tb927.2.6310, Tb927.5.300, Tb927.7.7500, and Tb09.211.4990. The deduced protein sequences contain a PTS1, suggesting a glycosomal localization. NanoLC-MS/MS, however, identified only two of these iron-ascorbate-oxidoreductases, Tb927.7.7500 and Tb927.5.300, in the glycosomes of PCF trypanosomes (Table 1J). Both proteins can be distinguished from the other iron-ascorbate-oxidoreductases by nanoLC-MS/MS for the presence of 1 unique peptide for Tb927.7.7500 and 1 unique peptide for Tb927.5.300. Iron-ascorbate-oxidoreductase (AOR) is a key-enzyme of the ascorbate-glutathione cycle, which in plant peroxisomes plays an important role in the detoxification of H2O2 [102]. During the peroxisomal ascorbate-glutathione cycle, reducing equivalents coming from glutathione or NADPH are transmitted via iron-ascorbate oxidoreductase to monodehydroascorbate, resulting in the formation of ascorbate (vitamin C), which subsequently reduces a peroxidase. The presence of two iron-ascorbate oxidoreductases in the PCF glycosome and the fact that trypanothione is able to spontaneously reduce monodehydroascorbate [103] suggests a similar mechanism for the removal of peroxides in the glycosomes of T. b. brucei (Fig. 2J). © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Intriguingly, also an NADP-dependent isocitrate-dehydrogenase (IDH: EC 1.1.1.42), containing a PTS1 sequence, was found in the glycosomes of both life forms (Table 1J). NADP-IDH catalyze the NADP-dependent reversible conversion of isocitrate to 2-ketoglutarate. In other eukaryotes, peroxisomal IDH provide NADPH for peroxisomal b-oxidation and glycerol-ether lipid biosynthesis and were shown to play an important role in the cellular defense against oxidative damage [104]. The presence of this enzyme in the PCF glycosome, and the lack of isocitrate- or 2-ketoglutaratemetabolising pathways, suggests a role for NADP-dependent IDH in the glycosomal defense against oxidative damage. NADPH-IDH would recycle NADP to NADPH required for the trypanothione-ascorbate cycle (Fig. 2J). This mechanism implies, in turn, the presence of a glycosomal isocitrate/2ketoglurate carrier, which has been proposed previously for peroxisomes [7, 105]. NanoLC-MS/MS revealed the presence of a putative isocitrate/2-ketoglurate carrier (Tb10.389.0690) in the glycosomes of PCF trypanosomes (see Section 3.17). 3.14 The mevalonate pathway The mevalonate pathway generates isoprenoid compounds, which are essential for many cellular functions [106]. The most abundant product of the mevalonate pathway is cholesterol, a constituent of membranes and the precursor for the biosynthesis of steroids. In eukaryotes, the initial steps of cholesterol biosynthesis occur in the cytosol, whereas the later steps take place in the ER. However, several enzymes of the cholesterol biosynthetic pathway have also been reported to exist in peroxisomes [107, 108]. The pathway starts with the synthesis of mevalonate from 3-hydroxy-3-methyl-glutaryl-CoA and is catalyzed by 3hydroxy-3-methyl-glutaryl-CoA reductase (HMGR, EC 1.1.1.34), a key enzyme that is subject to several regulatory mechanisms [106, 109, 110]. HMGR from T. b. brucei was reported to be microsomal [111], whereas Heise et al. [112] proposed a mitochondrial localization of this enzyme. The second step of the pathway, the conversion of mevalonate to mevalonate-5-phosphate, is catalyzed by mevalonate kinase (EC 2.7.1.36). BSF trypanosomes can only salvage cholesterol from the blood [113], whereas PCF trypanosomes can also synthesize ergosterol de novo [114]. Coppens et al. [111] showed that in PCF trypanosomes, the expression of HMGR is down-regulated in presence of cholesterol, which is normally present in the medium. As expected, we were able to detect mevalonate kinase, but could not detect HMGR in PCF glycosomes, confirming the cholesterol-dependent regulation of this enzyme in T. b. brucei (Table 1M). The next steps of the mevalonate pathway are catalyzed by diphosphomevalonate decarboxylase and phosphomevalonate kinase. Neither enzyme has a PTS1, and could not be detected in the T. b. brucei glycosomes by nanoLC-MS/MS, suggesting that further processing of mevalonate 5-phosphate most probably occurs in another subcellular compartment. www.proteomics-journal.com

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3.15 Threonine metabolism BSF and PCF trypanosomes were reported previously to be able to catabolize L-threonine [22, 115, 116]. Opperdoes et al. [52] showed that in PCF trypanosomes the threonine metabolism occurs exclusively in the mitochondrion: here threonine is converted to acetyl-CoA and glycine by the enzymes L-threonine 3-dehydrogenase (EC 1.1.1.103) and acetylCoA:glycine C-acetyltransferase (EC 2.3.1.29) [52]. BSF trypanosomes, however, lack a functional mitochondrion, and nothing was known so far about the subcellular localization of the threonine metabolism in this life form. NanoLC-MS/ MS analysis revealed the presence of L-threonine 3-dehydrogenase (Tb927.6.2790) in the glycosome of BSF trypanosomes (Table 1L). The protein sequence contains a PTS1, confirming its glycosomal localization. Why this enzyme is localized in the BSF glycosome is unclear.

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Tb09.160.4590 is present in the BSF glycosome remains unclear: not a single unique peptide was found for this arginine kinase. In the PCF glycosome, none of the mentioned arginine kinases could be detected (Table 1O). BSF trypanosomes contain a “non-functional” mitochondrion, and obtain their ATP exclusively by substratelevel phosphorylation during glycolysis [11, 16]. Previous reports have provided evidence that the glycosomal membrane is impermeable to several metabolites, and that the maintenance of the glycosomal ATP/ADP-ratio by a precisely balanced glycosomal metabolism is essential [25]. The presence of arginine kinases in BSF glycosomes, suggests the presence of an additional temporal energy buffer (argininephosphate) in these organelles, which would stabilize the ATP/ADP ratio and could provide energy under stress conditions. 3.17 Glycosomal membrane proteins

3.16 Phosphoarginine: A glycosomal high-energy phosphate buffer? Eukaryotic cells have evolved elaborate ATP-buffering systems based on phosphagen kinases, which maintain ATP homeostasis during high and fluctuating energy demands [117]. The common feature of all phosphagen kinases is their capability to synthesize large pools of ‘high-energy phosphates’, the phosphagens, during normal metabolic conditions and to replenish the ATP from this pool during periods of high-energetic demand [117]. Phosphagen kinase systems have several advantages compared to ATP as single energy source: phosphagens can accumulate to much higher intracellular concentrations as compared to ATP and diffuse faster due to their smaller size [118]. They provide further a temporal energy buffer that stabilizes cellular ATP/adenosine diphosphate (ADP) ratios and matches energy supply when the energy consumption becomes critical [118]. Phosphagens also trap a considerable amount of inorganic phosphate, which is liberated upon phosphagen hydrolysis and exerts regulatory effects on gluconeogenesis and glycolysis. Phosphoarginine is the main reserve of high-energy phosphate compounds in a wide variety of invertebrates, whereas in vertebrates only phosphocreatine is found [117]. Arginine kinases (EC 2.7.3.3) have been described previously in T. cruzi and T. b. brucei [119, 120], and were shown to be essential for trypanosome energy management during stress conditions [121]. The genome of T. b. brucei contains three different arginine kinase-encoding genes: Tb09.160.4560, Tb09.160.4570 and Tb09.160.4590. The proteins encoded by the last two genes are more homologous (94.3% amino acid identity) to each other then to Tb09.160.4560 (85% amino acid identity). So far, nothing was known about the subcellular localization of the different arginine kinases. NanoLC-MS/MS revealed the presence of two arginine kinases, Tb09.160.4560 and Tb09.160.4570, in BSF glycosomes (Table 1O): each could be distinguished from the other arginine kinases by a single unique peptide. Whether © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Previous reports have provided evidence that the glycosomal membrane and its protein constituents are trivial for glycosome biogenesis and parasite survival [27]. It was also shown that the glycosomal membrane is impermeable to several metabolites [25], implying the presence of specific transporters. Most of the metabolic pathways discussed above require co-participation of enzymes located in the glycosome and other cellular compartments. In addition, transporters are required for the maintenance of the redox balance in the glycosome. To get a better insight into glycosomal metabolism, and its rationale for the unique compartmentalization of glycolysis, identification of glycosomal membrane proteins is of key importance. PEX proteins (peroxins) act in different aspects of peroxisome biogenesis: import of matrix proteins, insertion of proteins into the peroxisomal membrane, recruitment of lipids for membrane formation, and the fission and inheritance of the peroxisomes. In T. b. brucei, only a small number of peroxins has been identified so far (see [32] and references therein). The glycosomal membrane proteins PEX11 and the related GIM5A/B are very abundant and were shown to be involved in glycosome division [30, 31]. The glycosomal membrane protein PEX2 [27] and PEX14 [122], and the soluble PTS1 receptor PEX5 [32] are involved in the import of glycosomal matrix proteins. NanoLC-MS/MS confirmed the presence of PEX11, GIM5A, PEX12 and PEX14 in both BSF and PCF glycosomes, whereas PEX2 was found exclusively in PCF glycosomes (Table 1Q). PEX2 is essential in both BSF and PCF trypanosomes, but the protein was reported previously to be undetectable in both life forms, suggesting that its abundance is very low [27]. This could explain our failure to detect PEX2 in BSF trypanosomes by nanoLC-MS/MS. Previous reports have provided evidence that the glycosomal membrane of T. b. brucei is impermeable to several metabolites [25], implying the presence of specific glycosomal metabolite transporters. The mitochondrial carrier family (MCF) was initially defined as a group of proteins that www.proteomics-journal.com

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are localized in the inner mitochondrial membrane, and mediate the transport or exchange of a wide range of metabolic intermediates [123–129]. Several structurally related carrier proteins have also been found in the membrane of peroxisomes from different eukaryotes [130–133]. NanoLC-MS/MS revealed the presence of three different MCF proteins in the glycosomes of PCF trypanosomes: Tb09.211.1750, Tb10.389.0690 and Tb10.61.1810 (Table 1Q). Tb09.211.1750 is homologous to mitochondrial phosphate carriers, which mediate the import of inorganic phosphate into the mitochondrial matrix either by proton co-transport or in exchange for hydroxyl ions [128]. This uptake of inorganic phosphate into mitochondria is essential for the phosphorylation of ADP to ATP. Additionally, inorganic phosphate participates in other phosphate-requiring reactions, and allows the uptake of various metabolites by exchanging inorganic phosphate via other carriers like the dicarboxylate carrier [128]. As discussed above, PCF trypanosomes contain a glycosomal pyrophosphate-dependent pyruvate phosphate dikinase, and it has been postulated that the equilibrium of this reaction is predominantly towards pyruvate, ATP and inorganic phosphate formation [76]. Accumulation of inorganic phosphate in the glycosome, however, would exert a negative regulatory effect on the ATP-consuming steps of glycolysis, suggesting that the glycosomal presence of this phosphate carrier is essential. Tb10.389.0690 is homologous to mitochondrial dicarboxylate carriers, which mediate the exchange of a wide variety of substrates, including dicarboxylates (2-ketoglutarate, fumarate, malate, succinate), tricarboxylates (isocitrate, citrate), inorganic phosphate and glutathione [134–136]. The presence of this metabolite carrier in the PCF glycosome was expected, because in this life form specific di- and tricarboxylates, but also glutathione, have to be exchanged across the glycosomal membrane. For example, glycolysis requires the exchange of fumarate and malate in order to maintain the glycosomal NADH balance (see Section 3.6), and the exchange of isocitrate and 2-ketoglutarate is essential for the maintenance of the glycosomal NADPH pool (see Section 3.13). The dicarboxylate carrier is also required for the exchange of oxidized and reduced trypanothione, which is required for the detoxification of peroxides (see Section 3.13). Tb10.61.1810 is highly homologous to mitochondrial ATP/ADP carriers. Previous reports have provided evidence that in the glycosomes of BSF trypanosomes the ATP/ADP balance is tightly regulated: there is no net ATP generation [25]. Consequently, there is no ATP/ADP carrier in BSF glycosomes (Table 1Q). For PCF trypanosomes, only limited information is available regarding the maintenance of the glycosomal ATP/ADP balance. Van Weelden et al. [20] showed recently that in PCF glycosomes more ATP is consumed than generated. A glycosomal ATP/ADP carrier, like Tb10.61.1810, could maintain the glycosomal ATP/ADP balance. However, mitochondrial ATP/ADP carriers are also the most abundant proteins found in the mitochondrial inner © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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membrane [137]. We can therefore not exclude a possible cross-contamination of our glycosome-enriched fractions with this carrier: further immunolocalization experiments will be required to confirm its glycosomal localization. NanoLC-MS/MS analysis revealed further the presence of the ABC transporter ABCD3 (Tb11.03.0030: Table 1Q), also called PMP70, in glycosomes of BSF trypanosomes. In the peroxisomal membrane of mammals, four ABC transporters have been identified, but the precise functions of these proteins is still unknown [138]. 3.18 Peroxisomes versus glycosomes Peroxisomes are present in almost every eukaryotic cell. They exhibit an exceptional functional versatility, varying considerably in their protein content with both the organism, the cell type in which they occur, and the environmental conditions to which the cells are exposed [3, 38, 105, 139, 140]. Like peroxisomes, glycosomes contain peroxins (PEX proteins) involved in organelle biogenesis, and were shown previously to be involved in the degradation of peroxides, the biosynthesis of ether lipids and cholesterol, and gluconeogenesis [5, 11]. Unique to glycosomes is the reported compartmentalization of glycolysis, the pentose phosphate pathway, the last steps of pyrimidine biosynthesis and purine salvage [5, 10, 11]. NanoLC-MS/MS analysis confirmed the presence of these proteins and pathways in the glycosomes (Table 1, Fig. 2). Hallmark of the peroxisome is the presence of the b-oxidation pathway, which is involved in the degradation of fatty acids [1, 105, 139]. Wiemer et al. [54] showed previously the presence of 2-enoyl coenzyme A hydratase and NADP-dependent 3-hydroxyacyl-CoA dehydrogenase enzyme activities in both the glycosomes and mitochondria of PCF T. b. brucei. However, activities of the other enzymes involved in this pathway, acyl-CoA synthase, acyl-CoA dehydrogenase, and 3ketoacyl thiolase, could not be detected. Scrutiny of the presented data indicated that the subcellular fractionation of PCF T. b. brucei (see Fig. 1 in [54]) was not optimal, and could have led to misinterpretation of the results. NanoLC-MS/MS analysis revealed clearly that none of the enzymes involved in b-oxidation is present in the glycosomes of both life forms. Our results imply that b-oxidation, the hallmark of peroxisomes, is apparently absent in glycosomes of T. b. brucei.

4

Concluding remarks

The “proteome” can be defined as the complement of proteins, encoded by a genome and transcriptome at a given point in time, which are performing enzymatic, regulatory, and structural functions. It is a highly dynamic entity that can change in response to the intracellular and extracellular environment: differences in culture conditions (medium, pH, temperature) as well as the cellular state (growth-phase at which the cells are harvested) will result in significant www.proteomics-journal.com

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changes of the protein complement. In addition, the accumulation of mutations in and adaptations of the laboratory strain used could result in significant changes of the proteome in time. These “limitations” of proteome profiling have to be kept in mind, especially when a comparison is made between proteomic (MS) data, such as those presented in this paper, and previously published experimental data. Moreover, there are additional limitations of which one should be aware. For example, it is sometimes not possible to determine by MS from which copy of a gene the identified protein was translated, as is demonstrated by glycerol kinase (see Section 3.4) and arginine kinase (see Section 3.16). Additionally, in a diploid organism, alleles of the same gene may slightly differ in their nucleotide sequence but only some allelic polymorphisms are represented in the genome sequence database. A good example here is the identification of PGKB in BSF glycosomes, which is clearly contradicting all of the experimental evidence published previously and is due to a single nucleotide difference between the two encoding alleles (Section 3.3). Another limitation is the known bias of the method for hydrophylic proteins, resulting in the identification of only part of the glycosomal membrane protein complement (see Section 3.17). Most membrane proteins are rather low abundant, and are barely digested by trypsin due to the presence of multiple transmembrane domains. In this report we provided for the first time a “snapshot” of the different biochemical processes taking place in the glycosomes of two replicating life forms of the kinetoplastid parasite T. b. brucei. When comparing the glycosomal proteomes of PCF and BSF trypanosomes, not only similarities but also remarkable differences were observed. Enzymes involved in glycolysis, purine salvage, pyrimidine biosynthesis, phospholipid degradation and glycerol-ether lipid biosynthesis were found in glycosomes of both life forms (Table 1, Fig. 2). PCF glycosomes, however, appear to be more complex and contain in addition enzymes of the oxidative branch of the pentose phosphate pathway, the CalvinBenson cycle and two different pathways involved in the detoxification of oxygen radicals and peroxides. Additional to known glycosomal enzymes and pathways, novel glycosomal constituents were identified by nanoLCMS/MS (Table 1, Fig. 2). New is the presence of enzymes involved in the Calvin-Benson cycle, the trypanothione- and trypanothione/ascorbate-dependent peroxide detoxification cycles, phosphagen biosynthesis by arginine kinase, and the recycling of NADPH by isocitrate dehydrogenase. Furthermore, several enzymes have been found that were not reported previously to be glycosomal, e.g. ribokinase, deoxyribosephosphate aldolase, phosphomannose isomerase, lysophospholipase, L-threonine 3-dehydrogenase and aldose 1-epimerase. To corroborate the glycosomal localization of the novel glycosomal constituents identified in this report, complementary experiments such as immunolocalization studies will be done. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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This research was financially supported through a grant (CL112/10) from the Deutsche Forschungsgemeinschaft (DFG). We thank Christine Clayton (ZMBH, Heidelberg, Germany) for support and the critical reading of the manuscript.

5

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