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BBAMCR-15465; No. of pages: 15; 4C: 3

Biochimica et Biophysica Acta xx (2006) xxx–xxx www.elsevier.com/locate/bbamcr

Metabolic functions of glycosomes in trypanosomatids ⁎ Paul A.M. Michels a, , Frédéric Bringaud b, Murielle Herman a, Véronique Hannaert a

a Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Université catholique de Louvain, ICP-TROP 74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium b Laboratoire de Génomique Fonctionnelle des Trypanosomatides, Université Victor Segalen Bordeaux 2, UMR-5162 CNRS, 146 rue Léo Saignat, 33076 Bordeaux cedex, France Received 30 April 2006; received in revised form 17 August 2006; accepted 18 August 2006

Abstract

Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several other processes such as the pentose-phosphate pathway, β-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would, through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions. © 2006 Elsevier B.V. All rights reserved.

Keywords: Trypanosome; Glycosome; Glycolysis; Differentiation; Metabolic adaptation; Organelle turnover

1. Introduction other organisms this metabolic process is essentially cytosolic. Glycosomes were subsequently also found in related protozoa Glycosomes were initially discovered in Trypanosoma (reviewed in [2–7]), all belonging to the Kinetoplastida, a group brucei [1], the parasite that causes human African sleeping that also comprises other parasites causing serious tropical sickness. These organelles appeared to contain most of the diseases in humans such as Trypanosoma cruzi and a variety of glycolytic enzymes, hence their designation as glycosomes. Leishmania species, as well as parasites of other vertebrates, Compartmentation of glycolysis within organelles is unique; in invertebrates and plants, and free-living organisms. These organelles belong to the peroxisome family, although initially some doubt existed about this affiliation because of the absence Abbreviations: ATG, autophagy-related gene; Gly3P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate; FH, fumarase; FRD, fumarate reductase; of catalase, the hallmark peroxisomal enzyme, from Trypano- GAT, glycosomal ABC transporter; GPO, glycerol-3-phosphate oxidase; HXK, soma and Leishmania glycosomes and the almost exclusively hexokinase; MCF, mitochondrial carrier family; PAT, peroxisomal ABC glycolytic role of the organelles in T. brucei; glycolytic transporter; PEP, phosphoenolpyruvate; PEPCK, PEP carboxykinase; PGK, enzymes comprise up to 90% of the glycosomal protein content phosphoglycerate kinase; PFK, phosphofructokinase; PPDK, pyruvate phos- when these parasites live in the mammalian bloodstream. Yet, phate dikinase; PTS, peroxisome-targeting signal; PYK, pyruvate kinase; TAO, trypanosome alternative oxidase; TCA, tricarboxylic acid like peroxisomes, these organelles are bounded by a single ⁎ Corresponding author. Tel.: +32 2 764 74 73; fax: +32 2 762 68 53. phospholipid bilayer, contain no DNA and catalase could be E-mail address: [email protected] (P.A.M. Michels). detected in glycosomes of some other kinetoplastids. Also

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Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

2 P.A.M. Michels et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx enzymes of ether-lipid metabolism are often present in different environments encountered by each of these differen- glycosomes [8,9], although not in those of bloodstream-form tiated forms. The mammalian-infective metacyclic form, that T. brucei. Furthermore, subsequent research showed that the finally develops in the salivary glands, is prepared again for an biogenesis of glycosomes occurs along similar routes as that of exclusively glycolytic metabolism in the mammalian blood. peroxisomes, and involves homologous proteins (peroxins) In contrast to T. brucei, which resides extracellularly (reviewed in [7]), and more recent biochemical studies as well throughout its life-cycle, T. cruzi and Leishmania species as genomic and proteomic analyses revealed more similarities at have additional, intracellular stages in their mammalian hosts: the metabolic level between glycosomes and other members of T. cruzi in the cytosol of various tissues, Leishmania in the the peroxisome family (see below, Sections 5, 6 and 7). lysosomes of macrophages. Consequently, their metabolism has to be able to rely also on the nutrients available in these highly 2. Glycosomes during the life-cycle of trypanosomatids specific environments, with consequences for the adaptability of their metabolism. Moreover, these parasites are transmitted Many Kinetoplastida have a complicated life-cycle [10,11]. by different insects (triatomine bugs and sand flies, respective- This holds particularly true for the best-studied kinetoplastid ly) and follow also different tracks within the insect vector. A organisms, the parasites belonging to the genera Trypanosoma need for metabolic adaptability to cope with different environ- and Leishmania. These organisms are transmitted between their ments is possibly also important for each of the many other, mammalian hosts by insects. Both in the mammal and the biochemically less well-characterized kinetoplastids irrespec- insect, they often transit through different parts of the body, tive whether they infect one or more specific hosts or are free each time being exposed to highly different environmental living. As will be argued below, glycosomes are likely to play conditions. Most transitions involve the cellular differentiation an important role in the metabolic adaptation to each of the of the parasite enabling it to adapt to the new environment environments to which kinetoplastid organisms are exposed. encountered, or to prepare itself for the next one. For example, T. brucei is introduced into the mammalian bloodstream by the 3. Compartmentation of glycolysis in glycosomes in bite of a tsetse fly. There it finds a largely constant, glucose-rich bloodstream-form T. brucei environment, allowing it to proliferate rapidly while producing all its ATP by a high rate of aerobic glycolysis or, at places When parasitizing the mammalian bloodstream, the African where the oxygen level is low, by anaerobic glycolysis. trypanosome T. brucei is, for its free-energy needs, totally Sequestering of the major part of the glycolytic pathway inside dependent on a continuous supply of glucose present in the glycosomes has been shown essential for glycolysis, ATP blood and body fluids of its host. All ATP synthesized by this production and growth of these bloodstream-form trypano- organism comes from the conversion of glucose into pyruvate, somes [12–14], and provides them with a unique mechanism the end-product of trypanosome glycolysis in this vertebrate for quickly switching from aerobic to anaerobic glycolysis (see stage. The seven enzymes involved in the conversion of glucose Section 3) [1,15,16]. This mechanism allows the trypanosomes into 3-phosphoglycerate are present inside the glycosome, while to overcome short periods of anaerobiosis, although it does not those catalyzing the last part of the pathway are localized in the enable them to synthesize sufficient ATP for sustaining growth cytosol (Fig. 1) [1]. The pyruvate is excreted into the host's [17]. Mitochondrial systems such as the tricarboxylic acid bloodstream. Within the glycosome, consumption and produc- (TCA) cycle, respiratory chain and the oxidative phosphoryla- tion of ATP and NADH by glycolysis are balanced: the tion machinery are largely repressed at this stage of the life- intraglycosomal consumption of ATP by hexokinase (HXK) cycle (reviewed in [3,18–20]). Such glycolysis-dependent, and phosphofructokinase (PFK) is compensated by the ATP long-slender trypanosomes are not prepared for life in the insect production by phosphoglycerate kinase (PGK). Net ATP midgut, where the concentration of sugars is usually very low formation occurs in the cytosol, in the reaction catalyzed by and that of amino acids, notably proline and threonine high; pyruvate kinase (PYK). Similarly, the glycosomal NADH therefore, they quickly die when ingested by a next fly taking a resulting from the reaction catalyzed by glyceraldehyde-3- meal. However, in the bloodstream the long-slender forms may phosphate dehydrogenase is re-oxidized inside the organelle differentiate into short-stumpy trypanosomes which are still followed by the transfer of the electrons to O2 via a glycolysis-dependent but have a partially de-repressed mito- mitochondrial glycerol-3-phosphate oxidase (GPO). This pro- chondrial system, allowing them to survive in the insect midgut. cess involves a glycosomal NADH-dependent glycerol-3- Upon ingestion, these short-stumpy trypanosomes differentiate phosphate dehydrogenase, a putative transporter (‘shuttle’)in rapidly into so-called procyclic forms with a much more the glycosomal membrane which exchanges glycerol 3- elaborate mitochondrial system, and also a considerably phosphate (Gly3P) for dihydroxyacetone phosphate (DHAP), different, more extensive glycosomal metabolic network (see and the mitochondrial GPO. The GPO is in fact a system Sections 4 and 5). In a period of 2–3 weeks, the trypanosomes comprising an FAD-linked glycerol-3-phosphate dehydrogen- move from the midgut, via the foregut and proboscis to the ase that is most likely associated with the outer surface of the salivary glands, while undergoing additional differentiation mitochondrial inner membrane, ubiquinone and a terminal steps resulting in morphologically distinct forms [21] which so oxidase, known as the trypanosome alternative oxidase (TAO). far have not yet been metabolically characterized. It is likely This respiratory process seems not to be involved in free-energy that the metabolic repertoire of the glycosomes varies with the transduction. The TAO is insensitive to cyanide, but can be

Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

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Fig. 1. Schematic representation of glycolysis in the bloodstream-form of T. brucei. Under aerobic conditions, glucose is converted into pyruvate. Under anaerobic conditions equimolar amounts of glycerol and pyruvate are produced. Abbreviations: 1,3BPGA, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; F-6-P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; G-3-P, glyceraldehyde 3-phosphate; G-6-P, glucose 6-phosphate; Gly-3-P, glycerol 3-phosphate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Pi, inorganic phosphate; UQ, ubiquinone pool. Enzymes are: 1, hexokinase: 2, glucose-6-phosphate isomerase; 3, phosphofructokinase; 4, aldolase; 5, triosephosphate isomerase; 6, glycerol-3-phosphate dehydrogenase; 7, glycerol kinase; 8, glyceraldehyde-3-phosphate dehydrogenase; 9, glycosomal phosphoglycerate kinase; 10, phosphoglycerate mutase; 11, enolase; 12, pyruvate kinase; 13, FAD-dependent glycerol-3-phosphate dehydrogenase; 14, alternative oxidase. inhibited by salicylhydroxamic acid. Inhibition of TAO mimics reaction, because now one molecule of 3-phosphoglycerate, the effect of a lack of oxygen on carbohydrate metabolism of the rather than two, is produced per molecule of glucose consumed. bloodstream form. Under these conditions, NAD+ is regenerated As a consequence, glucose is dismutated into equimolar by the reduction of DHAP to Gly3P, followed by the formation amounts of pyruvate and glycerol, with net synthesis of only of glycerol that is excreted by the organism. This is achieved by one molecule of ATP. reversal of the glycerol kinase reaction, thus synthesizing The regulation of glycolysis in trypanosomes differs largely glycerol and ATP from Gly3P and ADP [1,15,16]. However, this from that of other organisms. There is an apparent lack of reverse reaction is thermodynamically unfavourable and only activity regulation of the glycolytic enzymes HXK and PFK feasible by mass action at high Gly3P concentration and a high [22–24]. In most organisms, the activities of these two key ADP/ATP ratio, conditions that are difficult to attain in an entire enzymes are highly regulated by their products, by metabolites cell but may occur in a small, closed compartment. By this further downstream in the metabolism or by other (allosteric) reverse reaction, the ATP/ADP balance within the glycosomes is effectors [25]. This regulation serves two purposes. First, it maintained; the ATP formed in the glycerol kinase reaction prevents the loss of ATP by futile cycling when glycolysis and compensates for the loss of one molecule of ATP in the PGK gluconeogenesis occur simultaneously. Second, it has been

Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

4 P.A.M. Michels et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx argued, and experimentally confirmed by using Saccharomyces 4. Glycosomes and energy metabolism in insect-stage cerevisiae mutants, that the ‘turbo design’ of glycolysis requires T. brucei and other trypanosomatids a tight regulation of the first steps of glycolysis [26]. Indeed, absence of regulation may lead to unrestricted accumulation of During its journey through the tsetse fly, T. brucei undergoes glycolytic intermediates, a situation that will be highly toxic for several differentiation steps, adapting itself to each new the cell. environment encountered. The procyclic form living in the Proper compartmentation of glycolytic enzymes inside midgut is, with the bloodstream form, the only one that so far glycosomes and possession of intact organelles are essential could be axenically cultured and has thus been made amenable for the survival of bloodstream-form T. brucei. Indeed, several to biochemical investigations. Also the promastigote Leishma- experiments have provided evidence that mislocalization of nia spp. and epimastigote T. cruzi, which propagate in the glycolytic enzymes is deleterious for the parasite. Trypano- midgut of their respective insect vectors, are commonly studied somes contain two major isoenzymes of PGK located in in laboratories. Although the metabolic networks, both within different compartments of the cell: one (PGKg), present in the glycosomes and the fully developed mitochondrion, of all glycosomes, is only expressed in the mammalian bloodstream- these insect-stage trypanosomatids seem very similar, most of form parasites, while a cytosolic enzyme (PGKc) operates the recent data regarding glycosomes and energy metabolism predominantly in procyclic trypanosomes [27,28]. Blood- pertain to procyclic trypanosomes, since gene silencing by stream-form trypanosomes were rapidly killed when, from an RNAi could be extensively used in T. brucei [33,34], but failed additional ectopic gene, PGKc or a truncated form of PGKg to be functional in the other parasites [35,36]. without the glycosome-targeting signal (PTS1) was expressed When grown in commonly used glucose-rich media, [29]. Expression of an inactive PGKc had no effect, indicating procyclic T. brucei and epimastigote T. cruzi primarily use the that the toxicity depends both on enzyme activity and cytosolic glucose as carbon source [37,38], whereas amino acids location. Similar growth inhibition has been observed after (including L-proline) are the main energy source present in transfection of bloodstream-form T. brucei with the gene of S. their insect vector [39]. In glucose-depleted medium, however, cerevisiae triosephosphate isomerase expressed as a cytosolic procyclic trypanosomes considerably increase the rate of L- enzyme [17]. Furthermore, partial relocation of glycosomal proline consumption (6-fold), indicating that it is the major matrix proteins to the cytosol upon depletion of peroxins PEX2, carbon source used by the parasite in its natural environment 6, 10, 12 or 14 by RNA interference (RNAi) readily killed and glucose, when present, exerts a negative control on L- bloodstream-form cells [13,14,30,31]. proline metabolism [40]. Glucose is catabolized in the To understand why the presence of a glycolytic enzyme in glycosomes, as described above for the bloodstream-form the wrong compartment is detrimental for trypanosomes, one trypanosome (see Section 3), whereas L-proline degradation has to know the function or consequences of this form of takes place in the mitochondrion. Glycolysis appeared dispens- metabolic compartmentation. To this aim, a mathematical able, since procyclics of several strains have been successfully model of glycolysis in bloodstream-form T. brucei was adapted to glucose-depleted media with no significant effect on developed on the basis of kinetic data as determined for the growth rate [40,41]. Interesting is the non-essential role of different glycolytic enzymes [32]. One question addressed was glycosomes in procyclic trypanosomes grown in glucose- how the functional behaviour of trypanosome glycolysis would depleted media, as exemplified by the analysis of a PEX14 change if the pathway were not compartmentalized [12]. knock-down mutant [14]. Procyclic trypanosomes were killed According to the computer model this would not significantly by RNAi-dependent depletion of PEX14 only when glucose affect the steady-state glycolytic flux. But, strikingly, it will was present, suggesting that glycosomes are only essential to lead to toxic accumulation of hexose-phosphates upon addition compartmentalize a functional glycolytic pathway. This obser- of glucose. Such toxic accumulation is prevented by vation strengthens the hypothesis that glycosomes serve to compartmentation, since the kinases at the beginning of the prevent the dangerous effect of the turbo design of glycolysis pathway respond to the glycosomal ATP/ADP ratio, not the [12,26] (see Section 3). Experimental support for this cytosolic one. The glycosomal ATP/ADP ratio, which, hypothesis was provided by the observation that RNAi- according to the computer model, is usually low, controls the mediated knockdown of HXK rescued procyclic cells from activity of HXK and PFK and maintains the concentration of glucose toxicity, even though glycosomal proteins were the hexose-phosphates constrained within a narrow range. If mislocalized to the cytosol [42]. the enzymes would sense the higher cytosolic ATP/ADP ratio, The extensive tracheal system of insects renders all organs their activity would not be restrained and the intermediates and tissues, including the digestive tractus, highly aerobic [43]. would accumulate. Therefore, the computer analysis suggested Consequently, insect parasites have developed an aerobic that the sequestering of the glycolytic pathway within a metabolism, exemplified by the presence of two distinct mito- membrane that is poorly permeable for solutes is essential for chondrial terminal oxidases (the cyanide-sensitive cytochrome the parasite, because it compensates for the lack of activity oxidase and the salicylhydroxamic acid-sensitive TAO), both regulation of its enzymes. Indeed, experimental data have been using oxygen as electron sink. Indeed, the midgut trypanoso- obtained to support the notion that compartmentation of matids cannot adapt to long-term growth under anaerobic glycolysis serves such a regulatory function and will be conditions and go into reversible or irreversible metabolic arrest presented in Section 4. during anoxia [44–46]. Although they are dependent on oxygen

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for proliferation, the insect-stage trypanosomatids developed a [48–50]. The PEP-CO2 condensation catalyzed by PEPCK is fermentative-like metabolism, since glucose (and L-proline) is the initial step to the production, inside glycosomes, of converted into partially oxidized end-products (succinate, succinate, a major end-product excreted by most trypanosoma- acetate, lactate, ethanol and/or glycerol, depending on the tids [20,46,47,51,52]. Third, instead of being the final end- species) by so-called aerobic fermentation [47]. product, pyruvate is further converted into acetate, lactate and L- In these insect-stage trypanosomes too, glucose is essentially alanine in the mitochondrion and/or the cytosol [46,51,53,54]. metabolized inside the glycosomes (Fig. 2), but with three main The two former changes imply that the mechanism by with the differences compared with the bloodstream form of the parasite redox potential and ATP/ADP ratio are balanced within (for recent reviews see [5,18–20]). First, PGK is present in the glycosomes of procyclic trypanosomes differs from that in the cytosol and, therefore, 3-phosphoglycerate is produced in this bloodstream form of T. brucei. compartment [27]. Second, phosphoenolpyruvate (PEP) pro- The production of succinate from PEP involves two duced in the cytosol forms a metabolic branching point and is glycosomal NADH-dependent oxido-reductases, malate dehy- the substrate for two glycosomal kinases, i.e., PEP carbox- drogenase and fumarate reductase (FRDg) [49,51], which ykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) provide a means for the re-oxidation of NADH produced in

Fig. 2. Schematic representation of glucose metabolism in the procyclic form of T. brucei. Excreted end products of glucose metabolism (acetate, L-alanine, glycerol,

L-lactate, succinate and CO2) are in white characters on a black background. Arrows with different thicknesses tentatively represent the metabolic flux at each enzymatic step. Dashed arrows indicate steps which are supposed to occur at a background level or not at all. The mitochondrial outer membrane is permeable to metabolites and is only shown in the vicinity of the schematic electron-transport chain. Abbreviations: AA, amino acid; 1,3BPGA, 1,3-bisphosphoglycerate; C, cytochrome c; CoASH, coenzyme A; DHAP, dihydroxyacetone phosphate; F-6-P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; G-3-P, glyceraldehyde 3- phosphate; G-6-P, glucose 6-phosphate; Gly-3-P, glycerol 3-phosphate; OA, 2-oxoacid; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; SucCoA, succinyl-CoA; UQ, ubiquinone pool. Enzymes are: 1, hexokinase: 2, glucose-6-phosphate isomerase; 3, phosphofructokinase; 4, aldolase; 5, triosephosphate isomerase; 6, glycerol-3-phosphate dehydrogenase; 7, glycerol kinase; 8, glyceraldehyde-3-phosphate dehydrogenase; 9, glycosomal phosphoglycerate kinase; 10, cytosolic phosphoglycerate kinase; 11, phosphoglycerate mutase; 12, enolase; 13, pyruvate kinase; 14, phosphoenolpyruvate carboxykinase; 15, pyruvate phosphate dikinase; 16, glycosomal malate dehydrogenase; 17, cytosolic (and glycosomal) fumarase (FHc); 18, glycosomal NADH-dependent fumarate reductase; 19, mitochondrial fumarase (FHm); 20, mitochondrial NADH-dependent fumarate reductase; 21, glycosomal adenylate kinase; 22, malic enzyme; 23, unknown enzyme; 24, alanine aminotransferase; 25, pyruvate dehydrogenase complex; 26, acetate:succinate CoA-transferase; 27, unknown enzyme; 28, succinyl-CoA synthetase; 29, FAD-dependent glycerol-3-phosphate dehydrogenase; 30, rotenone-insensitive NADH dehydrogenase; 31, alternative oxidase; 32, F0F1-ATP synthase; I, II, III and IV, complexes of the respiratory chain.

Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

6 P.A.M. Michels et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx the organelle by the glycolytic glyceraldehyde-3-phosphate The relocation of PGK to the cytosol of trypanosomes when dehydrogenase. Theoretically, two NADH molecules are they differentiate from bloodstream to procyclic forms results consumed per molecule of succinate produced for a single from the expression of a different isoenzyme, PGKc instead of NADH molecule produced per molecule of PEP synthesized PGKg. This change has been interpreted in relation to the from glucose, implying that only half of PEP produced in the necessity to balance the ATP production and consumption cytosol needs to be re-routed to the organelle and converted into inside the glycosomes: in procyclics the phosphorylation of the succinate in order to balance the NADH/NAD+ ratio. However, glycolytically produced ADP would be performed by PEPCK the fraction of PEP metabolized in the glycosome is during the glycosomal succinic fermentation. However, the significantly higher than expected, since approximately 70% conversion of a significant part of PEP into pyruvate by the of the glucose consumed is converted into succinate [51,55]. cytosolic PYK implies that the PEPCK reaction would not This apparent discrepancy probably results from the existence contribute sufficiently to maintaining the glycosomal ATP/ADP of a second succinate production pathway, located in the balance. Consequently, at least another glycosomal ATP- mitochondrion, fed by malate produced in the glycosomes (Fig. producing step is required to complete the metabolic scheme. 2), which accounts for 14–44% of succinate produced from the This function may be fulfilled by a constitutively expressed glucose metabolism [52]. For each succinate molecule produced third PGK isoform, the glycosomal PGKA [27,28,61,62]. The in the mitochondrion, a single NADH molecule is formed in the dual localization of PGK activity (PGKA and PGKc) may help glycosomes, instead of two. Assuming that the glycosomal to maintain the ATP balance inside the glycosomes of procyclic succinic fermentation is sufficient for maintaining the glycoso- trypanosomes by altering the ratio of the fluxes from 1,3- mal redox balance, the Gly3P/DHAP shuttle described for the bisphosphoglycerate to 3-phosphoglycerate via the two differ- bloodstream-form T. brucei seems a superfluous pathway in the ent routes (Fig. 2). However, the contribution by PGKA to procyclic cells. However, also this insect form of the parasite glycosomal ATP production is debated, since its relative amount expresses a functional shuttle, providing additional metabolic is very low compared to the other isoforms in T. brucei, it has a flexibility that may be beneficial under particular growth low specific activity, and its activity is not detectable in conditions [56]. This is exemplified by the observation that glycosomal fractions of procyclic trypanosomes ([48], and FB, RNAi-dependent depletion of the glycosomal FRD activity unpublished data). The alternative of glycosomal ATP produc- does not affect growth rate while glucose is still consumed, tion by glycerol kinase is thermodynamically unfavoured, and albeit at a reduced level, suggesting that an alternative way thus unlikely, and indeed the procyclic form does not excrete (possibly the Gly3P/DHAP shuttle) is used by this mutant to significant amounts of glycerol when grown under standard substitute for NADH oxidation by FRDg [51]. conditions [46,47,51]. PPDK is another glycosomal ATP- Surprisingly, the glycosomal succinic fermentation pathway producing enzyme, which utilizes PEP, AMP and PPi to may make a detour to the cytosolic compartment (Fig. 2). Two produce pyruvate, ATP and Pi [50,63]. This PPi-dependent class I fumarases, respectively located in the cytosol (FHc) and enzyme was proposed to substitute for pyrophosphatase in order the mitochondrion (FHm), have recently been identified, to hydrolyze the PPi produced in the glycosomes by suggesting the absence of fumarase in the glycosomes [57]. biosynthetic pathways (see Section 5) which would otherwise Indeed, a recent proteomic analysis of glycosomes isolated be inhibited [4,5,50,63]. This hypothesis implies that the rate of from procyclic trypanosomes did not reveal a fumarase [58], ATP production by PPDK depends on the flux of the but contrasts with the previous detection of significant FH glycosomal PPi-generating pathways, which should be signif- activity in glycosomal fractions of procyclic trypanosomes icantly lower than the catabolic flux of glucose. Consequently, [51]. These contradictory data may be correlated to the the PPDK activity is probably not sufficient to maintain the presence of a cryptic PTS1 at the C-terminal extremity of T. glycosomal ATP/ADP balance, as confirmed experimentally, brucei FHc (AKLV). One could imagine that this cryptic PTS1 since the viability and glucose metabolism are not affected by becomes functional after a form of decrypting (by the activity PPDK gene knockout [38]. Alternatively, ATP could be of a carboxypeptidase) dependent on environmental condi- imported from outside the glycosomes, similar as reported for tions. A similar cryptic PTS1 is present at the C-terminal peroxisomes, where the yeast Ant1p and human PMP34 extremity of the orthologous T. cruzi FH (SKLL), whereas a nucleotide transporters are involved in the exchange of AMP- somewhat different sequence is found in the L. major enzyme ADP and ATP across the membrane, to provide intraperox- (SKTLA). In the latter case, but also for the Trypanosoma isomal ATP for β-oxidation or activation of fatty acids, species, alternative possibilities to explain the dual localization respectively [64,65]. Indeed, the recent proteomic analysis of are the existence of non-consensus targeting sequences or glycosomes from procyclic trypanosomes identified a protein piggy-back transport as discussed in more detail elsewhere [7]. homologous to mitochondrial ATP/ADP carriers, but a Nonetheless, whatever the import signal and mechanism, the mitochondrial contamination of the glycosomal-enriched frac- variability in the dual localization suggests a regulation of the tion cannot be excluded [58]. The presence of several ATP- import process. The unexpected cytosolic location of FHc may producing pathways in addition to a putative nucleotide be attributed to the requirement of fumarate (instead of NAD+) transporter may provide an important metabolic flexibility for as electron acceptor for the cytosolic dihydroorotate dehydro- adaptation to rapid environmental changes. However, questions genase, an essential enzyme of de novo pyrimidine biosyn- remain as to how the glycosomal ATP/ADP balance is thesis [59,60]. maintained in procyclic trypanosomatids.

Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

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Adaptive forms of all trypanosomatids analyzed to date, activity regulation mechanisms found in other organisms except the slender bloodstream form of T. brucei, express the [23,24] raise the still unsolved question as to how futile ATP NADH-dependent FRD activity [51,66–69] and produce hydrolysis resulting from the combined activity of these two significant amounts of excreted succinate from glucose glycosomal enzymes is avoided. [20,47]. Interestingly, succinate production seems to be Also another glucose consuming process, the oxidative correlated with the presence of a PGK activity in the cytosol, branch of the pentose-phosphate pathway, is compartmentalized since the bloodstream form of T. brucei is the only one lacking inside glycosomes [74,75]. Although most of the enzymes of the PGKc isoform. All other trypanosomatid forms analyzed this pathway in the three trypanosomatids for which the genome express at least one cytosolic PGK isoform, often simulta- sequencing has been completed appear to have a PTS, often a neously with glycosomal isoforms [27,28,70–72]. In other substantial part of their activity was found in the cytosol, words, the exclusively glycosomal localization of PGK is indicating only partial compartmentation; in T. cruzi the restricted to bloodstream-form T. brucei and correlates with the cytosolic contribution is even predominant [76]. Intriguingly, absence of the glycosomal succinic fermentation pathway. genes encoding a sedoheptulose-1,7-bisphosphatase with a Thus, the repression of the succinic fermentation pathway by PTS1 have been found in T. brucei and T. cruzi. This is a Calvin bloodstream-form T. brucei is accompanied with the repression cycle enzyme, usually only found in plastids of plants and algae of the cytosolic PGK in favour of the glycosomal isoform in and in some fungi and ciliates where it is possibly present in the order to balance the glycosomal ATP/ADP ratio. These cytosol exerting a still unknown function. Possibly the adaptations to the glucose-rich environment of the vertebrate trypanosomatids acquired the sedoheptulose-1,7-bisphospha- blood came along with the repression of the mitochondrial TCA tase by lateral gene transfer [73,77]; we hypothesize that the cycle enzymes and respiratory chain. Interestingly this parasites employ it in a modified pentose-phosphate pathway expression strategy appears to be a recent evolutionary (Fig. 3A), as described in more detail elsewhere [5]. adaptation by T. brucei, because it is not found in all other All trypanosomatids studied contain various isoforms of trypanosomatids including its closest known relative, T. adenylate kinase, an enzyme that is important for ATP congolense, which has a very similar life cycle; it also homeostasis. In T. brucei seven genes encoding the enzyme propagates in the blood of vertebrates after transmission by for several cell compartments have been found, including one tsetse flies. The bloodstream form of T. congolense excretes for a glycosomal enzyme [78]. The importance of this enzyme succinate and expresses both the glycosomal and cytosolic PGK for glycosomal metabolism has been shown by computer isoforms [47,70]. The T. congolense orthologous gene (c2PGK) simulation of metabolism and expression knockdown using of the T. brucei glycosomal PGKg gene encodes a cytosolic RNAi [32,78]. isoform. Consequently, PGKA is the only glycosomal isoform A glycosomal localization has previously also been reported expressed in T. congolense [70]. In conclusion, all trypanoso- for most enzymes of the purine salvage pathway in different matids analyzed so far developed the largely glycosomally- trypanosomatids and for a bifunctional enzyme, resulting from a located metabolic network for aerobic fermentation of glucose. gene fusion, catalyzing the last two of the six steps usually Only the long-slender bloodstream form of T. brucei could comprising the de novo pyrimidine biosynthetic pathway, afford to considerably reduce this network to the mere whereas the other four enzymes were found in the cytosol glycolytic pathway, possibly as a result of its constant and (Fig. 3B and C) (reviewed in [4,79]). Interestingly, the genes of glucose-rich environment. the enzymes for pyrimidine biosynthesis are organized in a highly unusual way for trypanosomatids, i.e., in an operon-like 5. Other metabolic systems in glycosomes manner, similar to that in cyanobacteria and not found elsewhere [80]. This organization and the sequence similarity Besides the glycolytic pathway and its extra branches strongly suggest that these genes were acquired by lateral gene towards glycerol and succinate production, many other transfer. As mammalian peroxisomes, trypanosomatid glyco- enzymes, often parts of pathways, have been found inside somes have been shown to contain the first two steps of ether- glycosomes during the last 20 years (Fig. 3). Most of these lipid biosynthesis, alkyl-DHAP synthase and DHAP acyltrans- enzymes were not, or only at very low activity, detectable in ferase (Fig. 3F) [8,9]. Also common with peroxisomes is the bloodstream-form T. brucei, but were shown to be present in association with glycosomes of some enzymes of β-oxidation glycosomes of cultured procyclic T. brucei and/or insect- or of fatty acids, such as 2-enoyl-CoA hydratase and an NADPH- mammalian-stage cells of Leishmania species or T. cruzi, dependent 3-hydroxyacyl-CoA dehydrogenase (Fig. 3E) mostly upon cell fractionation, sometimes by immunofluores- [81,82]. For T. brucei it was shown that these two enzymes cence studies with intact cells. And indeed most of the enzymes behave as a bifunctional enzyme [82]. were shown to possess a PTS1 or PTS2. For example, the key As mentioned above, the peroxisomal marker enzyme enzyme of the gluconeogenic pathway, fructose-1,6-bispho- catalase is absent from Trypanosoma and Leishmania species, sphatase, possesses peroxisome-targeting signals [73] and but it has been found in other kinetoplastids such as Crithidia indeed was demonstrated to be present in glycosomes of T. luciliae, Phytomonas sp. and Trypanoplasma borelli, where it brucei. The observation that this enzyme is expressed was located in the glycosomes [2,3]. But in recent years, a simultaneously with the glycolytic PFK (VH, unpublished different peroxide detoxification system has been unravelled result), and the apparent absence from trypanosomatid PFK of in trypanosomatids and components of it were found also in

Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

8 P.A.M. Michels et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx the glycosomes, in line with the possession of a PTS by The recently completed genome sequencing projects for T. some of them [83]. The unique trypanosomatid system is a brucei, T. cruzi and L. major [87] have allowed to get further cascade comprising a modified form of glutathione, N1,N8-bis insight into the metabolic repertoire of glycosomes by searching (glutathionyl)spermidine designated trypanothione, trypa- all protein sequences with either a potential C-terminal PTS1 or nothione reductase, a thioredoxin-related protein called try- an N-terminal PTS2 [80]. Moreover, proteomic analyses of paredoxin and 2-Cys-peroxiredoxin-type tryparedoxin glycosomes purified from both bloodstream- and procyclic- peroxidases and glutathione peroxidases (Fig. 3H) (reviewed form T. brucei have been initiated [58]. In the bioinformatic in [84]). Also other enzymes involved in oxidative stress analysis of L. major, 191 potential PTS1-containing proteins defence have been detected in glycosomes such as one of the and 68 potential PTS2-containing proteins with homologues in four iron-dependent superoxide dismutase isoenzymes of try- T. brucei and T. cruzi were identified. About 50% of these were panosomatids [85,86]. hypothetical proteins to which no function could be attributed as

Fig. 3. Overview of glycosomal metabolic processes other than glycolysis. Pathways or single reactions are depicted of which enzymes are probably at least in part sequestered in glycosomes of some life-cycle stages of T. brucei, T. cruzi and/or Leishmania species, as concluded from experiments involving cell fractionation in conjunction with activity assays, immunoblotting and/or mass-spectrometry based proteomics, or immunofluorescence microscopy of intact cells and/or from the identification of a (putative) peroxisome-targeting signal in the amino-acid sequence. The two circles given at each reaction indicate the probability of a glycosomal localization of the enzyme: the left circle signifies the availability of experimental support (filled circle) or the lack (so far) of such support (open circle); a filled right circle indicates the presence of a (potential) PTS, an open circle no detectable PTS. The pathways or processes indicated are: (A) pentose-phosphate pathway, (B) purine salvage, (C) pyrimidine biosynthesis, (D) energy homeostasis, (E) β-oxidation, (F) ether-lipid biosynthesis, (G) squalene synthesis, (H) oxidative-stress protection. In addition to the enzymes highlighted here many more candidate glycosomal enzymes and hypothetical proteins have been identified by genomic searches and proteomic analysis [80,58]. The sedoheptulose-1,7-bisphosphatase is proposed to connect the glycolytic and pentose-phosphate pathways, allowing to vary the yield for NADH and ATP from glycose metabolism according to the cells requirements, as discussed in more detail in [5]. For a more extensive discussion of the various reactions, the subcellular localization of the enzymes and their expression in the different trypanosomatids life-cycle stages, see the main text and references [4,5,58,80].

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Fig. 3 (continued). yet. The results of these genomic and proteomic studies largely precursors of many important compounds such as squalene confirm the earlier experimental findings described above, and eventually sterols (Fig. 3G). Three enzymes, responsible for while filling in some gaps in pathways (Fig. 3). However, the the conversion of mevalonate to 5-pyrophospho-mevalonate proteomic study did not detect any enzymes of β-oxidation, were each shown to possess a PTS in at least two of the three although two enzymes were found with a candidate PTS2 in all trypanosomatids. The same holds true for a fourth enzyme three trypanosomatids: the bifunctional 2-enoyl-CoA hydratase/ further in the pathway, squalene synthase, converting farnesyl- 3-hydroxyacyl-CoA dehydrogenase mentioned above and 3- pyrophosphate into squalene. The first enzyme of the pathway, ketoacyl-CoA thiolase. Possibly, the proteomic analysis was 3-hydroxy-3-methyl-glutaryl-CoA reductase had previously performed with trypanosomes cultured under conditions by been localized to mitochondrion and glycosomes [88]; its which the pathway was repressed. However, the bifunctional sequence has indeed a candidate mitochondrial transit peptide enzyme may also be targeted differently, since its sequence but not an identifiable PTS. The proteomic analysis detected the contains also a candidate mitochondrial transit peptide. mevalonate kinase only in glycosomes of procyclic cells. Additionally, the genome sequences of T. cruzi and L. major In addition to adenylate kinase mentioned above, glyco- revealed candidate glycosomal enzymes that should allow these somes may contain another enzyme involved in ATP homeo- parasites to feed the glycolytic pathway also with various other stasis, arginine kinase (Fig. 3H). T. brucei and T. cruzi, but not sugars than glucose [80]. Leishmania species, contain this enzyme; in T. brucei even The glycosome also harbours some enzymes of the three genes were identified coding for enzymes differing only in mevalonate pathway for the biosynthesis of isoprenoids, their N- and C-termini. One of them has a putative PTS1.

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Indeed, the proteomic analysis identified the enzyme in per molecule of glucose consumed. Any further decrease of glycosomes of bloodstream- but not procyclic-form trypano- this yield by spending ATP for transmembrane transport somes. Studies with T. cruzi have shown that phospho-arginine processes within the glycolytic process itself would be highly is particularly important during periods of stress; it is likely that disadvantageous for the cell, and thus unlikely. We therefore this phosphagen acts as an energy reserve during differentiation consider it more likely that other kinds of transport processes steps [89,90]. are responsible for the glycolytic intermediates. A first option is the involvement of MCF transporters. Indeed, trypanoso- 6. Metabolite transporters in the glycosomal membrane matid genomes contain several MCF homologues, most of them probably present in the inner mitochondrial membrane As for peroxisomes, several lines of evidence provide strong [87]. But the recent proteomic analysis detected three of them support for the notion that the membrane of glycosomes is in glycosome preparations of procyclic T. brucei [58]. How- poorly permeable (reviewed in [7]). First, the biphasic kinetics ever, none has been found so far in glycosome preparations of radioactive labelling of glycolytic intermediates, when from bloodstream-form cells. The three MCF molecules found trypanosomes received a pulse of 14C-glucose, suggested the in procyclic glycosomes present the highest percentage of existence of two pools of intermediates: a rapidly labelled identity/similarity to phosphate carriers, dicarboxylate carriers glycosomal one, representing 20–30% of the total pool of and ATP/ADP exchangers. However their functional identity glycolytic metabolites and a slowly labelled cytosolic pool remains to be determined and their glycosomal localization representing 70–80% of the intermediates [91]. Exchange confirmed. between both pools of intermediates appeared to happen only Several other possibilities for solute translocation through the very slowly. Second, under anaerobic conditions bloodstream- glycosomal membrane may be considered. (1) Entry into or exit form trypanosomes are still capable of performing glycolysis by from glycosomes by diffusion along the (electro)chemical producing one molecule of pyruvate and one molecule of gradient via specific transporters or pores not belonging to a glycerol per molecule of glucose consumed instead of two known class of proteins and that remain to be identified (2) molecules of pyruvate. As discussed in Section 3, this involves Exchange or antiport processes not involving MCF proteins, in the reversal of the glycerol kinase reaction, driven by which the import of one metabolite is obligatory coupled to the accumulation of Gly3P and a low ATP/ADP ratio as can only export of another one. Also in this case, the energy would be be achieved in a small, closed compartment separate from the provided by the transmembrane gradients of the compounds. cytosol. Further strong indications for the glycosomal mem- Kinetic and stoichiometric analyses suggest that such exchange brane as a permeability barrier are the above mentioned is very likely to happen for the transport of Gly3P and DHAP importance of maintaining a redox (NADH/NAD+) balance, [95,96]. Antiport systems for other metabolites could be the apparent requirement of stoichiometry for the sum of imagined as well. (3) A more speculative possibility is an reactions inside the organelle [32], and the observed detrimental association of substrate-specific soluble enzymes (in the effects resulting from mislocalization of glycolytic enzymes glycosomal matrix or cytosol) with non-specific carriers or [17,29]. pores in the glycosomal membrane, thus creating a kind of The existence of the permeability barrier implies the need for specific ‘group-translocation process’ by which the vectorial transporter molecules or pores for the exchange of specific process is driven by its coupling to the enzymatic conversion. substrates, products and intermediates. So far, two groups of This might, for example, be the case for glucose uptake and its candidate solute carriers, homologous to known transporters of subsequent phosphorylation by HXK, resembling the PEP- peroxisomal membranes [64,92] (see also elsewhere in this dependent phosphotransferase system responsible for glucose issue) have been identified in glycosomes: ABC transporters transport in many bacteria [97]. However the glycosomal system [93] and MCF proteins belonging to the Mitochondrial Carrier would be fundamentally different from the bacterial one, Family [58]. Three so-called half-ABC transporters (called because in trypanosomes glucose phosphorylation is performed GAT1-3, for Glycosomal ABC Transporter) have been by an authentic ATP-dependent HXK. Interestingly, trypanoso- identified in the glycosomal membrane of T. brucei, and matid HXK seems to have affinity for membranes. Furthermore, homologues were found in the genomes of T. cruzi and L. it could be imagined that also other enzymes may associate with major. T. brucei GAT2 has been shown to complement, to a such a non-specific pore or transporter, either other sugar-phos- limited extent, growth on oleate by Saccharomyces cerevisiae phorylating enzymes or enzymes catalyzing entirely different cells deficient in both peroxisomal fatty-acid transporters PAT1 reactions at the beginning or end of a glycosomal pathway. The and PAT2 [94]. GAT1 and GAT3 were not able to complement former option may be the case in glycosomes of the fish parasite the deficiency; no information is as yet available about their Trypanoplasma borreli, where the full set of glycolytic enzymes substrate specificity. GAT1 is predominantly expressed in could be found except HXK [98]. The fact that the metabolic procyclic T. brucei. Interestingly, GAT2 is only expressed in schemes presented above invoke many solute translocation steps bloodstream-form T. brucei in which glycolysis is by large the through the glycosomal membrane, together with the observed major function of glycosomes. However, for theoretical low protein content of the membranes and the difficulty in reasons, we consider it doubtful that ABC transporters are identifying such candidate transporters by classical biochemical involved in the transport of glycolytic intermediates. The ATP means and modern genomic and proteomic analyses seem to yield of glycolysis is low: maximally two molecules of ATP make it worth to experimentally pursue the latter option.

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7. Role of glycosomes in metabolic programming during pexophagy, a specific form of autophagy, as described in detail differentiation of trypanosomatids elsewhere in this issue (reviewed in [101,102]). Autophagy and pexophagy share several proteins with the cytoplasm-to- As described above, the life-cycle of trypanosomatids is vacuole targeting (Cvt) pathway by which some newly highly complex; the parasites are at every step morphologically synthesized vacuolar proteins of yeasts are routed to their and metabolically well adapted to a distinct compartment of organelles. This specific degradation of peroxisomes can occur their specific hosts. As the successive differentiation stages may by two different ways. The first one, known as micropex- encounter highly different environments, the parasites have to ophagy, involves the complete engulfment of the organelles by rapidly change their metabolism to cope with the altered extended vacuole arms. The second one, called macropexo- conditions. The compartmentation of environment-sensitive phagy is even more selective: the organelles to be degraded are parts of metabolism within glycosomes could be a way by surrounded by membranous layers to form an autophagosome, which the trypanosomes achieve such flexibility. As explained whose membrane will fuse with that of the vacuole. The in previous sections, the enzymatic content of the organelles induction of either micro- or macropexophagy depends on the may vary considerably from one adaptive form to the other; nature of a nutrient signal and differs between species. By the therefore, it could be imagined that a rapid way to adapt the combination of selective and induced degradation and synthesis cell's metabolism is changing its glycosome population by of peroxisomes, yeasts can create a population of organelles specifically destroying the old, not anymore adapted organelles with a different set of enzymes, and thus adapt efficiently and and inducing the synthesis of new ones, similar as has been rapidly to new environmental conditions. reported for yeast peroxisomes when growth conditions change The situation might be similar for trypanosomatids: the (Fig. 4). glycosomes may be heterogeneous; a subset of proliferation- The peroxisome renewal process has been studied in detail in competent organelles may give rise to a population with methylotrophic yeasts. Glucose-grown Hansenula polymorpha enzymes that allows differentiating parasites to deal with the contains only one or two small peroxisomes. When these cells new conditions encountered by the new life-cycle stage, are transferred to methanol-containing medium, proteins of whereas the old organelles could be degraded specifically. methanol assimilation are imported so the organelles grow and Indeed, recent evidence suggests the presence of autop- new organelles are formed by fission from the mature ones hagy processes in these organisms, particularly during the [99,100]. After fission the mature organelles lose the capacity to differentiation from one life-cycle stage into the next one. First, incorporate new protein; protein import is confined to the the occurrence of autophagy has been reported previously for L. smaller organelles that have budded off. The other part of the major [103,104], and our recent database searches using the process is the removal of the redundant organelles. The three trypanosomatid genomes against 40 autophagy-related degradation of peroxisomes occurs by a process called genes (ATG) coding for proteins involved in these processes

Fig. 4. Model of the turnover of glycosomes during different steps of the trypanosome's life cycle. For details, see text. Modified from [108].

Please cite this article as: Paul A.M. Michels et al., Metabolic functions of glycosomes in trypanosomatids, Biochimica et Biophysica Acta (2006), doi:10.1016/j. bbamcr.2006.08.019 ARTICLE IN PRESS

12 P.A.M. Michels et al. / Biochimica et Biophysica Acta xx (2006) xxx–xxx showed that the autophagy machinery is at least partially present unique in sequestering the glycolytic pathway, as well as some in these organisms [105]. In eukaryotic cells, the Cvt and other parts of the network for carbohydrate metabolism. This autophagy pathways can be divided in different steps including subcellular organization of metabolism appears to have some the induction and signalling, the cargo selection and packaging, important consequences for these organisms. Proper biogenesis the vesicle nucleation, the retrieval, the docking and fusion, and of glycosomes and correct glycosome-targeting of glycolytic finally, the vesicle breakdown [106]. Many of the proteins enzymes is essential for trypanosomes when grown on glucose, involved in these steps are implicated in different cellular probably because incorrect or incomplete compartmentation processes, others are specific for one or more forms of affects the regulation of the glycolysis. Moreover, the autophagy. For each of the different steps, orthologues were compartmentation enables these organisms to overcome short either confidently identified in at least one trypanosomatid periods of anaerobiosis, albeit without sustaining growth. database or were found to be likely present. Only the Cvt Interestingly, the enzymatic content of glycosomes may vary pathway seemed absent. However, for each step of autophagy importantly between different life-cycle stages of parasitic only half of the proteins were identified, suggesting that the trypanosomatids. Possibly, the sequestering of their pathways autophagy process in trypanosomatids is much simpler than that for carbohydrate metabolism within glycosomes enables the of yeast, but still viable from a genomic point of view since no parasites to adapt rapidly to new nutritional conditions during key compounds were absent. Recently, autophagy was found to the differentiation of one life-cycle stage to the next by the ‘en play a role in the differentiation and acquisition of the virulence bloc’ degradation of one population of organelles through of L. major [107]. An ATG4 null mutant was unable to autophagy, concomitant with the synthesis of a new population differentiate into the infective form. Using a fluorescent ATG8 of glycosomes with a different enzymatic content. This fusion protein as an autophagosome marker, an increase of glycosome-dependent capacity for efficient and rapid metabolic autophagy was monitored during differentiation. These results adaptation may have played an important role in the successful confirmed the presence of these two proteins, which were both radiation of kinetoplastids as parasites with multiple, highly classified as ‘likely to be present’ in the genomic survey, as well different niches in one or several host species. as the presence of a specific autophagy structure, the autophagosomes, in trypanosomatids. Acknowledgements Furthermore, our genomic analysis seemed to indicate that the parasites possess the molecular machinery to degrade The research by PM and VH was supported through grants selectively their glycosomes by a process analogous to either from the ‘Fonds de la Recherche Scientifique Médicale’ (FRSM) macro- or micropexophagy. This analysis needs now to be of the ‘Communauté française de Belgique’ and the Interuni- confirmed experimentally. Our (MH and PM) unpublished versity Attraction Poles-Belgian Federal Office for Scientific, results with T. brucei have revealed that, during the differen- Technical and Cultural Affairs. The research by FB received tiation of this parasite from the bloodstream to the procyclic support from the CNRS, the Conseil Régional d'Aquitaine and form, glycosomes co-localize with enlarged lysosomes and the Ministère de l'Education Nationale de la Recherche et de la glycosomal enzymes specific for the bloodstream form Technologie. MH acknowledges a PhD scholarship from the disappear rapidly after differentiation, concomitant with the ‘Fonds pour la formation à la Recherche dans l'Industrie et dans synthesis of specific procyclic ones. l'Agriculture’ (FRIA) and the Université catholique de Louvain. 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