biogenesis and function of peroxisomes and glycosomes

Molecular & Biochemical Parasitology 115 (2001) 19–28

Reviews: Parasite Cell Biology: 2

Biogenesis and function of peroxisomes and glycosomes

Marilyn Parsons a,b,*, Tetsuya Furuya a,b, Sampa Pal a,b, Peter Kessler a,b

a Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98177, USAb Department of Pathobiology, School of Public Health and Community Medicine, Uni�ersity of Washington, Seattle, WA 98195, USA

Received 13 November 2000; received in revised form 27 February 2001; accepted 5 March 2001

Abstract

Peroxisomes of higher eukaryotes, glycosomes of kinetoplastids, and glyoxysomes of plants are related microbody organellesthat perform differing metabolic functions tailored to their cellular environments. The close evolutionary relationship of theseorganelles is most clearly evidenced by the conservation of proteins involved in matrix protein import and biogenesis. Theglycosome can be viewed as an offshoot of the peroxisomal lineage with additional metabolic functions, specifically glycolysis andpurine salvage. Within the parasitic protozoa, only kinetoplastids have been conclusively demonstrated to possess glycosomes orindeed any peroxisome-like organelle. The importance of glycosomal pathways and their compartmentation emphasizes thepotential of the glycosome and glycosomal proteins as drug targets. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Trypanosoma ; Leishmania ; Glycolysis; Protein import

www.parasitology-online.com.

1. Introduction

Glycosomes, peroxisomes, and glyoxysomes are re-lated organelles that lack a genome and are bounded bya single membrane. These three types of microbodiesexist in different cellular environments and possessdistinct specialized functions. This review will provide asummary of our understanding of the biogenesis of thisgroup of microbodies, including the molecules involvedin import of matrix proteins and the targeting of mem-brane proteins. Additionally, we will describe selectedfeatures of glycosomal pathways that are distinct fromthose in mammalian peroxisomes, or that may providea possible target for chemotherapeutic intervention.Due to space limitations, referencing is representativerather than exhaustive, and focuses on more recentcontributions.

Table 1 outlines the metabolic pathways or enzymesfound in each class of microbodies — clearly there aresimilarities and significant differences. Catalase is con-sidered to be a hallmark of peroxisomes. Trypanosomesand Leishmania apparently lack this enzyme [1]. On theother hand, glycosomes, which are found in kinetoplas-tid parasites, are unique in that they house a part of theglycolytic pathway [2]. Glyoxysomes, present in green-ing plants, are so named because they contain theglyoxylate cycle. All three types of organelles containthe enzymes for �-oxidation of fatty acids. The overlap-ping compartmentation, combined with conservation ofprotein import processes (see below), support the con-tention that glycosomes and glyoxysomes can be con-sidered as variants of the prototypic peroxisome.Differences between peroxisomes, glyoxysomes, andglycosomes may have resulted from differential reten-tion of pathways from the ancestral peroxisome [3] orfrom retention of core activities supplemented by differ-ential relocation of pathways to the organelle [4].

In most organisms, peroxisomes are not essential forcellular survival. Peroxisome-deficient yeast cells cansurvive on most carbon sources [5], and mammaliancells without peroxisomes can survive in tissue culture.

Abbre�iations: mPTS, membrane protein peroxisomal targeting se-quence; PEX, peroxin; PMP, peroxisomal membrane protein; PTS,peroxisomal targeting sequence.

* Corresponding author. Tel.: +206-284-8846/315; fax: +206-284-0313.

E-mail address: [email protected] (M. Parsons).

0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0166-6851(01)00261-4

M. Parsons et al. / Molecular & Biochemical Parasitology 115 (2001) 19–2820

Table 1Pathways and enzymes of peroxisomes and glycosomes

Yeast peroxisomes Glyoxysomes GlycosomesMammalian peroxisomes

�-oxidation of fatty acids Yes Yes Yes YesYes YesYes Species dependentCatalase

YesEther lipid synthesis No No YesNoGlycolysis No No Yes

Yes YesNo NoGlyoxylate cycleYesIsoprenoid biosynthesis No ? ?

No No YesPurine salvage No

However, genetic defects in peroxisomal biogenesis orfunction in humans generally result in death in infancyor childhood [6]. Conversely, proliferation of perox-isomes is associated with hepatocellular carcinoma [7].In mammals, peroxisome proliferation is triggered byexposure to certain xenobiotics that activate transcrip-tion of genes involved in peroxisomal function [7].

Among parasitic protozoa, clear evidence for glyco-somes or peroxisomes is restricted to the Kinetoplas-tida, including the suborders Trypanosomatina(Trypanosoma, Leishmania, Crithidia, and Phytomonas)[1] and Bodonina (Trypanoplasma, Cryptobia) [8,9]. Intrypanosomatids, there appears to be no analog of theperoxisome proliferation seen in mammals. Perox-isomes have not been described ultrastructurally forany other parasites. Indeed, there is no evidence for thepresence of these organelles in Giardia, Trichomonas, orEntamoeba. The presence of peroxisomes in Apicom-plexa is the subject of current debate [10,11].

The information we describe in this review comesfrom several different systems. Two deserve specialmention. First, the generation and analysis of mutantyeast defective in peroxisomal function or biogenesishas been invaluable. Similarly, studies of fibroblastsfrom patients with peroxisomal disorders have providedinsights into mammalian peroxisome biogenesis. Whilethe contributions of parasitology have been more lim-ited in the area of biogenesis and protein import, theyhave served to identify many common features of theseprocesses. In addition, they have illuminated some in-teresting features unique to the glycosome.

2. Overview: building the peroxisome

The process of forming a functional peroxisome in-volves multiple pathways. Lipids must be recruited toform the organellar membrane. Little is known aboutthis process, although it has been demonstrated that thelipid composition of the peroxisomal and glycosomalmembrane differs from that of the total cellular profile[12]. The import of matrix proteins requires a set ofperoxisomal proteins as well as a cytosolic receptor.This pathway is distinct from that required for insertion

of proteins into peroxisomal membranes. Several spe-cies of yeast have served as important model organismsto study the proteins involved in building a functionalperoxisome [5]. In these organisms, peroxisomes areinduced by growth on specific nutrients such asmethanol or oleic acid, but are not essential for growthon standard media. Among the mutants defective ingrowth on these substrates are cells with defects inperoxisomal biogenesis. Such mutants fall into twomajor classes: those with peroxisomal ghosts (emptyperoxisomes which nonetheless contain peroxisomalmembrane proteins (PMPs)) and those that have nodetectable peroxisomal structures. Genetic complemen-tation of those mutants has allowed the identification ofthe relevant genes. The corresponding proteins aretermed peroxins, abbreviated as PEX. Most PEX genesare moderately conserved, with putative homologs be-ing present in organisms as diverse as S. cere�isiae,humans, and trypanosomatids. For a comprehensivereview of protein import into the peroxisome mem-brane and matrix, please see [13].

3. Targeting of matrix proteins

Peroxisomal and glycosomal matrix proteins are syn-thesized on free ribosomes in the cytosol and thentransferred to the organelle, typically with no prote-olytic modification. Three classes of topogenic signalshave been identified for peroxisomal matrix proteins.The best defined of these is the C-terminal targetingtripeptide Ser-Lys-Leu (or a related sequence) [14,15].This motif, termed the peroxisomal targeting sequencetype 1 (PTS1), is present on most peroxisomal matrixproteins. Many glycosomal proteins end in a PTS1-likesequence and typically this region is required for glyco-somal targeting. Differences in the acceptable degener-acy of the signals for trypanosomes and mammalianperoxisomes have been noted [15]. Nevertheless, thesedata strongly suggested that the mechanism of importof peroxisomal and glycosomal proteins would sharecommon features. This hypothesis was strengthened bythe identification of a second type of PTS (PTS2),which is present near the N-terminus of several perox-

M. Parsons et al. / Molecular & Biochemical Parasitology 115 (2001) 19–28 21

isomal proteins [16]. A similar sequence found on try-panosome aldolase is important for targeting of theprotein to the glycosome in vivo [17]. In addition tothese motifs, there are internal targeting sequenceswhich still remain to be defined [18,19]. In trypanoso-matids, some enzymes are encoded by a single gene, yetare partitioned between glycosome and cytosol [4].Studies of targeting of reporter proteins have shownthat changes in the PTS1 tripeptide [15], as well asalterations in adjacent sequences [20], can cause duallocalization, probably as a result of lowered affinity forthe PTS1 receptor. Interestingly, a catalase gene clonedfrom T. gondii encodes a protein with a PTS1 [10,11].The localization of the encoded protein, however, iscontroversial and as such cannot be considered clearevidence for the organelle in T. gondii.

4. Import of matrix proteins

One class of PEX genes encode peroxins that areinvolved in the import of matrix proteins. When thesegenes are disrupted, functional peroxisomes are notformed, but peroxisomal ghosts are observed. As withother organelles, protein import into peroxisomes in-volves multiple proteins, including those that reside instable complexes and those that interact in a moretransitory manner. Known interactions, most of whichare conserved among species, are summarized in Fig. 1.Fig. 2 is a cartoon featuring key molecules as thatfunction in protein import into the peroxisomal matrix.Many PEX proteins contain motifs for protein-proteininteraction, including tetratricopeptide (TPR) motifs,WD repeats, SH3 domains, and cysteine rings, as sum-marized in Table 2. Interestingly, none of the PEXproteins appear to be homologous to proteins involvedin mitochondrial or chloroplast protein import.

The first step in peroxisomal protein import is theinteraction of the targeting sequence with a receptor.PTSs are first recognized in the cytosol. Indeed, boththe PTS1 receptor, PEX5, and the PTS2 receptor,PEX7, appear to cycle between cytosol and peroxisome[21–23]. Defects in the corresponding genes result inthe absence of the respective PTS1 or PTS2 proteins inthe organelle. The mislocalized proteins are generallyfound in the cytosol, although some proteins appear tobe degraded if not localized to the proper organellarenvironment [24].

PEX5 homologs have been identified in many organ-isms including mammals, yeasts, L. dono�ani [25] andT. brucei [26]. PTS1 interactions with PEX5 have beenmapped to the seven tetratricopeptide repeats (TPRs)located in the carboxy portion of the protein. TPRs aredegenerate 34 amino acid repeat units present onproteins involved in diverse cellular functions. The crys-tal structure of TPR domains reveals a helix-turn-helix

motif with interacting anti-parallel �-helices [27].Protein–protein interactions can be accommodated onboth the inner and outer face of the super helix, sug-gesting that the PEX5 TPR interface could interactsimultaneously with multiple proteins.

The binding of PEX7 to peroxisomal matrix proteinsis PTS2 dependent [28]. All PEX7 protein homologshave WD motifs, a 44–60 aa sequence named for itsconserved Trp-Asp di-peptide. WD repeats form a �propeller fold [29]. As with the TPR repeats of PEX5,the WD structure is not predicted to provide enzymaticactivity, but rather a structure that coordinates thesimultaneous interaction of several proteins [29]. In thisway both PEX5 and PEX7, after recognition of therespective PTSs, are poised to interact with dockingpartners at the peroxisomal membrane. Interestingly, inmammalian cells but not yeast, PEX5 interacts withPEX7 and is required for the import of PTS2 proteins[30]. Whether trypansomatids resemble mammals oryeasts in this regard is not yet known.

A number of integral and peripheral PMPs functionin the import process and disruption of the correspond-ing genes typically leads to defects in import of bothPTS1 and PTS2 proteins (Fig. 2). Among such proteinsare those of the docking complex, which contains (atminimum) the integral membrane protein PEX13 plusthe peripheral membrane proteins PEX14 and PEX17[21,31–34]. The cytosolically oriented SH3 domain ofPEX13 mediates the interaction with PEX5 [35], whilePEX14 and PEX17 interact directly [33,34], possiblythrough their coiled coil motifs. These coiled coils serveas dimerization domains in several families of proteins[35,36].

In addition to the docking complex, multiple PEXproteins thought to participate in the import cycle havebeen identified. PEX2, PEX10 and PEX12 are integralPMPs that contain RING fingers. RING fingers, likezinc fingers, bind two molecules of zinc [37]. PEX5accumulates on peroxisomes in PEX2, PEX10 andPEX12 mutants [22,38], suggesting that these RINGfinger proteins function downstream of the dockingstep. These data also suggest the presence of a specificPEX5 recycling mechanism. It is not yet clear whetherthe PTS receptors are internalized prior to recycling.

Exactly how bound proteins traverse the peroxisomalmembrane remains unclear. Surprisingly, proteinslocked in a folded conformation by chemical cross-link-ing or stabilizing drugs can be imported into perox-isomes and glycosomes [39,40]. Moreover, proteinslacking a PTS can be imported into peroxisomes if theyassociate with a protein bearing a PTS [41,42]. Thesedata suggest that, in contrast to mitochondrial andchloroplast protein import, proteins may be importedinto peroxisomes in a folded or multimeric state.


Fig. 1. Network of PEX protein interactions. Open ovals represent soluble proteins; shaded proteins represent peripheral and integral membraneproteins. Most PEX interactions have been demonstrated by both yeast two-hybrid analysis and co-immunoprecipitation or pull downexperiments. In general, PEX proteins that interact in one species also interact in other species. The exception appears to be PEX7, whichapparently shows different interactions in yeast (PEX7/14 and PEX7/13) and mammalian cells (PEX7/PEX5). Indirect interactions cannot be ruledout in all cases.Fig. 2. Cartoon of key molecules involved in peroxisomal protein import. The cytosolic receptors bound to their cognate PTSs are shownapproaching the docking complex at the peroxisomal membrane surface. On the side are the RING proteins, whose exact relationship with theimport complex remains unknown. Lines and complementary shapes indicate protein interactions (see also Fig. 1). Additional PEX proteins notshown in this diagram are likely to be involved in protein import as well.

5. The peroxisomal membrane

Compared with matrix protein targeting, little isknown about the targeting of PMPs. PMPs do not use

PTS1 and PTS2 sequences for targeting to the peroxiso-mal membrane. PMP47 has six predicted transmem-brane domains. The membrane protein PTS (mPTS)has been mapped to a 20 amino acid hydrophilic loop


between transmembrane domains 4 and 5. This regionis both necessary and sufficient for (peripheral) associa-tion with the peroxisomal membrane [43]. PEX3, aPMP essential for peroxisomal biogenesis, has a singletransmembrane domain and its N-terminus is luminal.The N-terminal 33 aa of human PEX3, which containsthe transmembrane domain, is necessary and sufficientfor integration of a reporter protein in the peroxisomalmembrane [44]. Sequence comparisons of PEX3 andPMP47 from several species has identified a small clus-ter of basic amino acids that are conserved in theregions identified as required for peroxisomal targeting[45]. PEX15 has a single transmembrane domain; itsC-terminus extends into the peroxisomal matrix [46].Proper targeting of this PMP has been mapped to theC-terminal 82 amino acids, which contains thetransmembrane domain and a small cluster of basicamino acids similar to that seen PEX3. Thus, while themPTS sequence requirements remain only a rough out-line, a cluster of basic amino acids that ultimately residewithin the peroxisomal matrix is likely to be involved.

Only three PEX knockouts lack peroxisomes andperoxisomal ghosts: PEX3 [47], PEX16 [48], andPEX19 [47]. The corresponding proteins, therefore,may be involved in the biogenesis of the peroxisomalmembrane. PEX16 is an integral membrane protein,but little is known about its function. Overexpression ofPEX3 leads to multiplication of peroxisomes, suggest-ing a role in peroxisomal membrane synthesis. PEX19is predominantly cytosolic. It interacts with numerousPMPs, including PEX3 [49,50]. This finding led to thesuggestion that PEX19 functions as a receptor formPTSs [50]. However, the regions of PMPs required forperoxisomal localization often do not overlap with theregions required for PEX19 binding, which is incompat-ible with the hypothesis described above [49].

The role of the ER in peroxisomal biogenesis contin-ues to be the subject of much debate. The juxtapositionof ER and peroxisomal membranes in electron micro-graphs led to the hypothesis that peroxisomes might bederived from the ER. This hypothesis fell into disfavoras it was recognized that many peroxisomal matrix andmembrane proteins are made in the cytosol and trans-ported to the peroxisome directly [51]. Nonetheless,intriguing studies in the yeast Yarrowia lipolytica point

to ER involvement in peroxisome biogenesis in thisspecies. For example, Y. lipolytica mutants defective inthe secretory pathway show altered trafficking of PEX2and PEX16 and disruption of certain PEX genes affectssecretion [52]. Evidence for trafficking of PMPsthrough the ER has been equivocal. In some reports,disruption of ER to Golgi trafficking has interferedwith trafficking of certain PMPs [53,54], while otherreports find the opposite result [48,55]. Arguing againsttrafficking of PMPs through the ER are studies on pex3or pex19 knockouts in human and S. cere�isae. In thesecells, which lack even peroxisomal ghosts, PMPs arenot associated with the ER, but are cytosolically de-graded or are associated with mitochondria [47,50,55].Furthermore, introduction of a PEX19 protein engi-neered to contain a nuclear localization signal into apex19 knockout strain, leads to the nuclear localizationof several PMPs [50].

Two members of the AAA protein family, PEX1 andPEX6, have been implicated in peroxisome biogenesis.AAA proteins are ATPases associated with diversecellular activities. Many AAA proteins have beenshown to participate in vesicular fusion events. Muta-tions in PEX1 or PEX6 lead to loss of peroxisomalfunction, and the appearance of small vesicles contain-ing PMPs and a low level of matrix proteins. PEX1 andPEX6 interact with one another and may function infusion of pre-peroxisomal vesicles [56]. Another set ofdata suggests these molecules act late in peroxisomalmatrix protein import [57].

6. PEX genes in parasitic protozoa

Analysis of the Plasmodium falciparum genome data-base, which is nearly complete, has not revealed thepresence of PEX genes [11]. These preliminary dataargue against the presence of the peroxisome in P.falciparum, and perhaps other Apicomplexa, althoughit should be noted that many of these genes are nothighly conserved from organism to organism.

In contrast, several PEX genes have been identified intrypanosomatids. These genes have been identifiedthrough functional screens (PEX2) [58], protein purifi-cation and gene cloning (PEX11) [59] or by virtue of

Table 2Interaction domains in PEX proteins

Protein Interaction module Structure Target

PEX5 PTS1Multiple 34 amino acid repeat unitsTPRWD �-propeller foldPEX7 PTS2

PEX13 SH3 Anti-parallel � sheets PEX14, PEX5PEX RINGsCross brace structure binding zinc atomsRING fingerPEX2, PEX10, PEX12PEX14, PEX17?Heptad repeats forming a left handed superhelixCoiled coilPEX14, PEX17


homology with PEX sequences in other organisms(PEX5) [25,26]. Additional potential homologs can beseen in the genome databases. Thus far, no viable nullmutants for any trypanosomatid PEX genes have beenreported, despite several targeted attempts [58,59]. Ourlaboratory has also used chemical mutagenesis to gen-erate multiple independent mutants that show partialdefects in glycosomal protein compartmentation [86].Again, no null mutants were obtained. These data pointto the essential nature of the glycosome in the parasiteand contrast with the fact that peroxisomes are gener-ally not required for cellular survival in other organ-isms. Such data support the concept that blocking theglycosomal compartmentation process may be of thera-peutic potential. However, no specific inhibitors of PEXprotein function in any organism have been identified.As the structures of these proteins are further defined,such an approach may be possible.

7. Metabolic functions and potential drug targets

7.1. Glycolysis

The presence of glycolytic enzymes for the conver-sion of glucose into 3-phosphoglycerate is the hallmarkthat distinguishes the glycosome from the peroxisome[2]. There is no net ATP synthesis within the glyco-some, but the 3-phosphoglycerate generated within theorganelle is metabolized further in the cytosol, generat-ing ATP through substrate-level phosphorylation. Theregeneration of the reducing equivalents necessary forglycolysis is accomplished through a glycerophosphateshuttle between the glycosome and mitochondrion [1].

Glycosomal glycolytic enzymes show stage-specificchanges in abundance, with T. brucei showing the mostdramatic changes. Procyclic forms generate energythrough cytochrome-mediated respiration while blood-stream forms lack this pathway and generate energythrough glycolysis. Correspondingly, the levels of glyco-somal glycolytic enzymes are much higher in blood-stream forms than procyclic forms [60]. Hence,bloodstream stage T. brucei have been a good model tostudy the function of glycolytic compartmentation. T.brucei bloodstream forms have a very high flux rate ofglycolysis [1]. Since the glycosomal membrane is aphysical barrier to the products and coenzymes inglycolysis, it was speculated that compartmentationsupported this high flux rate by limiting diffusion of thesubstrates. However, later studies based on calculationsof the theoretical flux rate suggested that even withoutglycosomal compartmentation the diffusion of the sub-strates should not limit glycolysis [61].

Alternative functions for the glycosomal compart-mentation of glycolysis have been suggested by morerecent studies. Expression of the last glycosomal gly-

colytic enzyme, phosphoglycerate kinase, in the cytosolis toxic to the bloodstream form of T. brucei [62]. Thistoxicity is dose-dependent and also dependent on theactivity and cytosolic localization of the enzyme. Thesedata suggest that one function of compartmentation isto circumvent metabolic interference.

Mathematical models based on characterization ofpurified glycolytic enzymes support this contention.Such models have been used to simulate the effect ofglycosomal compartmentation in T. brucei bloodstreamforms [61]. The studies showed that the presence of aglycosome membrane had little effect on the steady-state flux rate. However, the model indicated that gly-cosomal compartmentation avoids osmotic effects whenextracellular glucose is high. Compartmentation alsowas critical in recovery from starvation; the closedglycosomal system would prevent the consumption ofATP, allowing glycolysis to restart when substrateswere available. Thus, the compartmentation appearsmost important when regulation of the pathway iscritical. Indeed, earlier studies have shown that theglycolytic enzymes typically involved in regulating thepathway in other organisms do not possess similarregulatory properties in T. brucei [1].

The mathematical model based on the kinetic datafor each enzyme was used to calculate which step in thepathway could be the most effective target to inhibit theglycolytic flux in T. brucei [63]. The plasma membraneglucose transporter was identified as the most pro-mising target, followed by the glycosomal enzymesaldolase, glycerol-3-phosphate dehydrogenase, glycer-aldehyde-3-phosphate dehydrogenase, and phospho-glycerate kinase [63]. In addition to the kinetic models,structural analysis indicates that several of the parasiteenzymes may be useful drug targets. For example, theintertwined PTS2 sequences on aldolase subunitspresent a unique structure that might be a target fordisruption of glycolysis [64]. Another promising candi-date is the recently crystallized glycerol-3-phosphatedehydrogenase. This enzyme, which is inhibited by theanti-parasitic agent melarsoprol, shows extensive differ-ences from the mammalian enzyme [65]. Recently, in-hibitors of trypanosomatid glyceraldehyde-3-phosphatedehydrogenase were designed based on the enzymestructure [66]. These compounds were toxic to T. bruceiand T. cruzi in vitro.

7.2. �-oxidation of fatty acids

In most organisms, peroxisomes are the site of oxida-tion of long, very long, and branched chain fatty acids,while mitochondrial enzymes oxidize medium and longchain fatty acids [67]. The products of �-oxidation inthe mitochondrion (acetyl CoA, NADH, FADH) arefed into the Krebs cycle and electron transport chain togenerate ATP. The peroxisomally generated products


(acetyl CoA, NADH and peroxide) are not directlycoupled to an energy generating system. It is thoughtthat the acetyl-CoA may be used in peroxisomal iso-prenoid and ether lipid biosynthesis [67]. Compartmen-tation of reactions with a high potential for oxidativedamage, such as cytochrome-mediated respiration andphotosynthesis, may serve to protect the cell fromperipheral damage. The same rationale can be appliedto compartmentation of �-oxidation of fatty acids,which involves repeated rounds of oxidation and thegeneration of peroxide.

Peroxisomes are impermeable to molecules such asNADH and acetyl-CoA [68]. Therefore, it is generallybelieved that peroxisomes and glycosomes haveproteins within their membrane that transport the nec-essary substrates and products. Several ABC trans-porter cassettes have been identified in peroxisomes(e.g. PMP70, ALDRP in humans; Pat1p, Pat2p in S.cere�isae) and at least some of these molecules functionin the transport of activated fatty acids [69]. PEX11,which is not an ABC transporter, is thought to partici-pate in the transport of fatty acids or a necessarycofactor for �-oxidation, although the mechanism of itsaction is unclear [70].

The �-oxidation pathway appears to be involved inthe regulation of peroxisomal abundance. Human cellsdeficient in enzymes mediating �-oxidation show a de-creased number of peroxisomes [71]. PEX11 is alsoinvolved in regulation of peroxisome size and abun-dance. Interestingly, despite the lack of evidence forglycosome proliferation in response to environmentalagents, overexpression of T. brucei PEX11 leads to adramatic increase in the size of glycosomes [59].

7.3. Ether lipid biosynthesis

Ether lipids (alkoxyphospholipids) are characterizedby the presence of an ether linkage in lieu of the typicalacyl linkage. The enzymes of ether lipid biosynthesis arefound within mammalian peroxisomes and trypanoso-matid glycosomes. Ether lipid synthesis does not occurin plants and yeasts. Cell fractionation has demon-strated that the enzymes for the first three steps in etherlipid biosynthesis are glycosomal in T. brucei andLeishmania mexicana [72]. Glycosomal ether lipid syn-thesis proceeds from dihydroxyacetone phosphate(DHAP) plus acetyl-CoA [72]. Protease protection ex-periments suggest that the first two enzymes, DHAPacyltransferase (which possesses a PTS1), and alkyl-DHAP synthase reside within the peroxisome matrix,the third enzyme, acyl/alkyl-DHAP reductase, likelyfaces the cytosol [73]. The alkyl-glycerol-3-phosphatethus generated is available for utilization in variouscellular processes.

In trypanosomatids, ether lipids have important rolesin the association of major surface molecules with the

plasma membrane through glycosylphosphatidylinositol(GPI) anchors [74]. While most proteins, including thevariant surface glycoproteins of Trypanosoma brucei,use GPI anchors that only contain ester lipids, the GPIanchor of Leishmania promastigote lipophosphoglycan(LPG) contains the unusual ether lipid 1-O-alkyl-2-lyso-phosphatidylinositol [75]. Another set of majorsurface molecules, glycoinositol phospholipids, havelinkages to the plasma membrane through anotherether lipid, either alkylacyl-phosphatidylinositol orlyso-1-O-alkyl-phosphatidylinositol [76]. Thus, etherlipids are likely to have important roles in Leishmaniaspp.

Some ether lipid analogues used in anti-cancer treat-ment are also effective against Leishmania parasites[77]. Hexadecylphosphocholine (miltefosine), a syn-thetic phospholipid without a glycerol backbone) hasshown promising activity in humans visceral leishma-niasis [78]. Although the mechanism for the anti-Leish-mania activity of miltefosine is not yet clear, these datasuggest the importance of ether lipids in Leishmaniaand the potential of the corresponding biosyntheticpathway as a drug target.

7.4. Isoprenoid synthesis

Two general pathways for isoprenoid synthesis havebeen described; one that is plastidic and mevalonate-in-dependent, and one that is peroxiosomal and meval-onate-dependent. Mevalonate is synthesized usingacetyl-CoA for the carbon backbone, and then is con-verted to farnesyl diphosphate (FPP) within the perox-isome. FPP is important not only as a precursor fordolichol, sterols, heme A and ubiquinone, but also forthe farnesylation of cytoplasmic proteins [79]. In mam-malian cells, the five enzymes that convert mevalonateto FPP are predominantly peroxisomal [79]. In Try-panosoma cruzi, the activity of 3-hydroxy-3-methyl-glu-taryl-CoA (HMG-CoA) reductase, which catalyzessynthesis of mevalonate, was reported as predominantlyglycosomal [80]. In contrast, the enzyme appears to bemitochondrial in T. brucei [81]. The localization ofdownstream enzymes remain to be determined. Severalisoprenoid derivatives inhibit the growth of T. cruziepimastigotes [82]. Inhibitors of sterol biosynthesis alsorepress parasite growth [83].

7.5. Purine sal�age

Trypanosomatids lack pathways for de novo synthe-sis of purines and rely completely upon purine salvagefrom the host. Surprisingly, several purine salvage en-zymes, which are cytosolic in all other eukaryotes, arelocalized exclusively to the glycosome. Those character-ized thus far include hypoxanthine:guanine phosphori-bosyl transferase, adenine phosphoribosyl transferase


and xanthine phosphoribosyl transferase [84,85]. Eachof these enzymes contains a PTS1 sequence at theC-terminus. The dependence of the parasites on purinesalvage makes these enzymes of interest for therapeuticintervention. Indeed, these enzymes are poorly con-served in trypanosomatids. However, null mutants forindividual enzymes are viable, due to the ability tointerconvert purines [85]. Thus, a therapeutic attackwill require the use of subversive substrates or targetingof multiple enzymes.

The role of compartmentation in microbodies ap-pears to vary with the pathway involved. Avoidance ofcellular oxidative damage and metabolic interferenceare some of the dangers that compartmentation canobviate. While many aspects of microbody biogenesisare more easily studied in yeasts or mammalian cells,the unique presence of diverse pathways in glycosomesmay offer new insights into the roles of compartmenta-tion and offer opportunities for therapeuticintervention.

Acknowledgements

This work was supported in part by grant AI31077from the NIH. Dr Pal is supported by Fogarty D43TW00924, International Training and Research inEmerging Infectious Diseases.

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