Báo cáo khoa học: Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis docx

In all mammalian cells capable of de novo synthesis of phos-pholipids, PC is generated by the CDP-choline path-way, often referred to as the CDP-choline branch of the Kennedy pathway [14

Trypanosoma brucei: a model micro-organism to study

eukaryotic phospholipid biosynthesis

Mauro Serricchio and Peter Bu¨tikofer

Institute of Biochemistry and Molecular Medicine, University of Bern, Switzerland

Introduction

Trypanosoma bruceiis a eukaryotic protozoan parasite

causing African sleeping sickness in humans and

nagana in domestic animals During its complex life

cycle, it migrates between the blood and tissue fluids

of a mammalian host and several compartments of the

insect vector, the tsetse fly Trypanosoma brucei is not

only a devastating pathogen, affecting social and

eco-nomic development in sub-Saharan Africa, but has

also become an interesting model organism to study

general biological processes RNA editing [1], glycosyl-phosphatidylinositol (GPI) anchoring [2], trans-splicing [3] and antigenic variation [4] represent biological phe-nomena that were initially discovered in trypanosomes and have later been observed in other eukaryotic organisms as well The T brucei genome with  9000 protein-coding genes has been sequenced [5] and the parasite is amenable to reverse genetic approaches, such as gene knockout by homologous recombination,

Keywords

biosynthesis; eukaryote;

glycerophospholipid; phospholipid; RNAi;

sphingophospholipid; trypanosome

Correspondence

P Bu¨tikofer, Institute of Biochemistry

& Molecular Medicine, University of Bern,

Bu¨hlstrasse 28, 3012 Bern, Switzerland

Fax: +41 31 631 3737

Tel: +41 31 631 4113

E-mail: peter.buetikofer@mci.unibe.ch

Website: http://ntbiomol.unibe.ch/Buetikofer/

(Received 12 November 2010, revised 23

December 2010, accepted 7 January 2011)

doi:10.1111/j.1742-4658.2011.08012.x

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of

de novo synthesis of all major phospholipids Here we provide an over-view of the biosynthetic pathways involved in phospholipid formation in

T brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis We show that de novo synthesis

of phosphatidylethanolamine involving CDP-activated intermediates is essential in T brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggest-ing a role for phosphatidylcholine formation in the control of cell division Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T brucei and review the biosynthesis of phosphati-dylinositol, which seems to take place in two separate compartments Finally, we emphasize that T brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inosi-tolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated

Abbreviations

CEPT, CDP-choline ⁄ ethanolamine:diacylglycerol phosphotransferase; CL, cardiolipin; CPT, CDP-choline:diacylglycerol phosphotransferase;

CT, CTP:phosphocholine cytidylyltransferase; EPC, ethanolaminephosphorylceramide; EPT, CDP-ethanolamine:diacylglycerol

phosphotransferase; ER, endoplasmic reticulum; ET, CTP:phosphoethanolamine cytidylyltransferase; GPI, glycosylphosphatidylinositol; IPC, inositolphosphorylceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol;

PGP, phosphatidylglycerophosphate; PI, phosphatidylinositol; PS, phosphatidylserine; RNAi, RNA interference; SM, sphingomyelin.

or RNA interference (RNAi)-mediated downregulation

of gene expression In addition, factors required for

adaptation and growth of different life cycle forms

cannot only be investigated in cell culture, but also

in suitable animal models (tsetse flies, rodents),

allow-ing host–pathogen interactions to be studied (reviewed

in [6]) Furthermore, in vitro differentiation of T brucei

from bloodstream- to procyclic (insect)-form parasites

may reveal changes in gene expression and metabolism

that are essential for the parasite life cycle (reviewed in

[7]) Interestingly, T brucei and other flagellates of the

order Kinetoplastida contain single (e.g mitochondria)

and unique (e.g glycosomes) organelles that undergo

dramatic functional and morphological changes during

differentiation, making T brucei an interesting model

organism to study organelle biogenesis and turnover

(reviewed in [8,9]), and cell division (reviewed in [10])

It has long been known that T brucei bloodstream

forms acquire lipids from their mammalian hosts For

this reason, the study of lipid biosynthesis in

trypano-somes has received little attention in the past Only

recently, a series of reports demonstrated that both

bloodstream- and insect (procyclic)-form parasites are

capable of de novo synthesis of lipids (recently

reviewed in [11]) Identification of eukaryotic routes

for lipid biosynthesis and of novel, parasite-typical

pathways raised a new interest in T brucei as a model

organism to study eukaryotic lipid homeostasis This

review provides an overview of the biosynthetic

path-ways for phospholipid synthesis in T brucei and

high-lights differences and unique features that may make

trypanosomes an attractive model micro-organism to

study lipid turnover and lipid–protein interactions in

eukaryotes

Biosynthesis of phosphatidylcholine

(PC)

PC represents the most abundant glycerophospholipid

class in most eukaryotes (reviewed in [12,13]) In all

mammalian cells capable of de novo synthesis of

phos-pholipids, PC is generated by the CDP-choline

path-way, often referred to as the CDP-choline branch of

the Kennedy pathway [14] It involves the sequential

action of three enzymes to generate PC from its

pre-cursors, choline and diradylglycerol, via the

high-energy intermediate CDP-choline Although in most

mammalian cells this pathway is responsible for the

production of almost the entire pool of PC (reviewed

in [15]), human liver cells synthesize approximately

one-third of their PC via sequential methylation of

phosphatidylethanolamine (PE) [16], a reaction

cata-lysed by PE N-methyltransferase [17] Both PC

biosynthetic pathways are involved in the regulation of lipoprotein metabolism in mice [18,19] However, they generate distinct pools of PC consisting of different molecular species [20] A similar observation has also been reported in the yeast, Saccharomyces cerevisiae [21] Very recently, the lack of PE N-methyltransferase was shown to protect mice against diet-induced obesity and insulin resistance [22], suggesting that this path-way may be linked to the regulation of body energy metabolism

All three enzymes involved in PC formation via the CDP-choline branch of the Kennedy pathway have been identified and characterized in mammalian cells The cytosolic enzyme choline kinase catalyses the first step in the reaction sequence, phosphorylating choline

in an ATP-dependent reaction to phosphocholine Choline kinases are ubiquitously distributed among eukaryotes [23] and, in general, use both choline and ethanolamine as substrates (reviewed in [24]) In mam-malian cells, choline kinase exists as three different isoforms encoded by two separate genes Recent studies suggest an important role for choline kinase in cancer cell proliferation (reviewed in [25]) The second enzyme in the CDP-choline pathway, CTP:phospho-choline cytidylyltransferase (CT), uses phosphoCTP:phospho-choline and CTP as substrates to form CDP-choline, thereby releasing pyrophosphate In mammalian cells, several isoforms of the enzyme have been described (reviewed

in [15,26]), consisting of up to four distinct conserved domains [23,27] Upon stimulation by lipids, CT is converted from a soluble to a membrane-bound form (reviewed in [28]) In many cells, CT has been localized

to the nucleus, but cytosolic forms of the enzyme have also been reported [15] The reaction catalysed by CT

is considered the rate-limiting step in PC synthesis In the final step of the CDP-choline pathway, a choline phosphotransferase activity transfers phosphocholine from CDP-choline to diradylglycerol to yield PC, releasing CMP as by-product Two different enzymes catalysing this reaction were identified and character-ized in mammalian cells, a CDP-choline⁄ ethanol-amine:diacylglycerol phosphotransferase (CEPT) that uses both CDP-choline and CDP-ethanolamine as sub-strates [29] and a CDP-choline:diacylglycerol phospho-transferase (CPT) that uses CDP-choline only as the substrate [30] Both CEPT and CPT are predicted to

be integral membrane proteins and have been reported

to localize to the endoplasmic reticulum (ER)⁄ nuclear membrane and Golgi, respectively [31] CEPT and CPT activities have also been identified in S cerevisiae [32,33]

In T brucei, candidate genes encoding all enzymes

of the CDP-choline pathway have been identified

(reviewed in [11]) Choline kinase, which in contrast

to mammalian cells is encoded by a single gene in

T brucei[23], has been characterized experimentally in

bloodstream-form trypanosomes and displays dual

specificity for choline and ethanolamine, with choline

being the preferred substrate [34], thus reflecting the

situation in most mammalian cells [26] The second

enzyme of the CDP-choline pathway, CT, has not been

studied experimentally in T brucei A recent report

suggests that part of the substrate for CT,

phospho-choline, may derive from sphingomyelin (SM)

degrada-tion by neutral sphingomyelinase [35] Based on the

importance of the first two enzymes of the

CDP-cho-line branch of the Kennedy pathway in mammalian

cells and yeast, they are probably essential in T brucei,

but experimental evidence is lacking Interestingly, all

kinetoplastid CTs are unusual fusion proteins in

hav-ing a cytidylyltransferase domain fused to a

CDP-alco-hol phosphatidyltransferase domain that is normally

found in CEPT and CDP-ethanolamine:diacylglycerol

phosphotransferase (EPT) [23] The function of this

additional domain is unknown The third enzyme,

CEPT, has been characterized in T brucei procyclic

forms and is involved in the synthesis of both PC and

PE Ablation of CEPT activity using RNAi caused a

reduction in PC and PE levels and a growth arrest of

parasites in culture [36] (Fig 1) Subsequent flow

cytometry and cytology studies demonstrated that

knocking-down CEPT expression inhibits nuclear

divi-sion [37], suggesting, for the first time in a eukaryotic

organism, a role for CEPT in the control of cell

divi-sion Although at present there is no information

available on the localization of CEPT in trypano-somes, its involvement in nuclear division suggests that

it may associate with the nuclear envelope membrane

in T brucei parasites, which would be consistent with its localization in mammalian cells [31]

In contrast to mammalian cells and yeast, the

T brucei genome lacks a candidate gene for PE N-methyltransferase Accordingly, experiments in both bloodstream [38] and procyclic forms [36] have shown that methylation of PE to PC does not occur in

T brucei

Biosynthesis of PE and phosphatidylserine (PS)

PE generally represents the second major glycero-phospholipid class in eukaryotes, whereas PS occurs in small amounts only (reviewed in [13,39]) Apart from being a major structural component of eukaryotic and prokaryotic membranes, PE has been shown to affect protein folding [40] and promote membrane fusion and fission events [41] In addition, PE can serve as a mem-brane anchor for proteins [42], and represents the donor of the ethanolamine moiety for GPI anchor bio-synthesis [43] and the ethanolamine phosphoglycerol modification of eukaryotic elongation factor 1A [44]

It is worth mentioning that the dependence of GPI and ethanolamine phosphoglycerol synthesis on PE as the ethanolamine donor was demonstrated using

T brucei as the model organism, although these pro-tein modifications had been reported for the first time

in other eukaryotic organisms

Fig 1 PE and PC formation in T brucei (A) In T brucei, EPT catalyses the final reaction in alk-1-enyl-acyl PE formation by the Kennedy pathway, whereas the dual-specificity enzyme, CEPT, generates diacyl PE and PC (B) RNAi-mediated ablation of EPT results in a reduction

in alk-1-enyl-acyl PE species and an accumulation of diacyl PE and PC (C) RNAi-mediated knockdown of CEPT results in a reduction in PC and diacyl PE species and a small increase in alk-1-enyl-acyl PE Changes in the PE and PC contents in (B) and (C) relative to control cells (A) are reflected by the sizes of the circles and the numbers The morphological and biochemical changes caused by RNAi are indicated at the bottom.

The biosynthesis and turnover of the two

amino-phospholipids PS and PE is metabolically closely

inter-related In mammalian cells, PS is synthesized via head

group exchange with an existing phospholipid, i.e by

replacing the choline group of PC or the ethanolamine

group of PE with the amino acid, l-serine The

reac-tions are catalysed by two distinct activities, PS

syn-thase-1 and PS synthase-2, showing different substrate

specificities for PC and PE, respectively [45,46] Both

enzymes are localized to mitochondria-associated

membranes, i.e special subdomains of the ER that

transiently come in contact with mitochondrial outer

membranes [47] However, the different tissue

distribu-tion of the two enzymes suggests that they may have

different functions (reviewed in [39]) Knockout mice

for PS synthase-1 or PS synthase-2 are viable and

exhi-bit minor phenotypes only [48–50], indicating that the

two enzymes may have complementary functions in

the maintenance of PS homeostasis

In contrast, S cerevisiae generates PS from

CDP-diacylglycerol and l-serine by the action of PS

synthe-tase [51], a membrane protein localizing to a special

subfraction of microsomes [52] A similar reaction

involving a membrane-associated enzyme also occurs

in Gram-positive bacteria [53] However, in

Gram-neg-ative bacteria, PS is synthesized by a cytosolic enzyme,

which only associates with membranes upon

interac-tion with lipid substrates [54], suggesting that

Gram-positive and Gram-negative enzymes evolved from

different origins (reviewed in [55]) Whereas PS

synthe-tase from the Gram-positive bacterium Bacillus subtilis

shows 35% overall amino acid sequence homology

to S cerevisiae PS synthetase, with particularly high

homology in the conserved CDP-alcohol

phosphatidyl-transferase domain, it shows little homology to PS

syn-thetase from Escherichia coli [56] Interestingly,

conserved amino acid motifs in E coli PS synthetase

indicate that it belongs to a large superfamily of

pro-teins that includes PS synthetases of other

Gram-nega-tive bacteria, bacterial cardiolipin (CL) synthases,

phospholipases D, nucleases and pox envelope proteins

[57]

PS is not only a membrane component and mediates

important cellular functions (reviewed in [58]), but also

serves as the substrate for PE formation In most

bac-teria, conversion of PS to PE, a reaction catalysed by

PS decarboxylase, represents the only pathway for PE

synthesis (reviewed in [59]) Similarly, in yeast and

many mammalian cells, decarboxylation of PS is a

major pathway for PE formation (reviewed in

[39,60,61]) Eukaryotic PS decarboxylases belong to

two distinct classes of enzyme that localize to different

intracellular compartments Type I PS decarboxylases

are found in mitochondria, whereas type II enzymes localize to the endomembrane system Typically, PS decarboxylases are membrane proteins consisting of two nonidentical subunits that are generated from sin-gle proenzymes The contribution of PS decarboxyl-ation to cellular PE formdecarboxyl-ation varies between different cell types or organisms A PS decarboxylase knockout

in mice results in mitochondrial defects and lethality between days 8 and 10 of embryonic development [62]

In eukaryotes, PE can also be synthesized via the CDP-ethanolamine branch of the Kennedy pathway [14], by head group exchange with PS, or by acylation

of lyso-PE (reviewed in [58]) The contributions of the latter two pathways to de novo synthesis of PE are unclear The first reaction of the CDP-ethanolamine branch of the Kennedy pathway is catalysed by etha-nolamine kinase, resulting in the formation of phosphoethanolamine, which in turn is activated using CTP by CTP:phosphoethanolamine cytidylyltransfer-ase (ET) to form CDP-ethanolamine Alternatively, phosphoethanolamine may also be produced via degra-dation of phosphate by sphingosine-1-phosphate lyase [63] The contribution of this reaction

to de novo PE formation in mammalian cells has not been firmly established The final step in PE formation

by the Kennedy pathway is catalysed by the dual-spec-ificity enzyme CEPT, transferring the ethanolamine moiety to diradylglycerol Interestingly, it has long been thought that CEPT provides all of the ethanol-amine phosphotransferase activity for PE formation However, recently, a human cDNA encoding a CDP-ethanolamine-specific phosphotransferase (EPT) was isolated and its transcripts were found ubiquitously expressed in multiple tissues [64]

The contribution of the PS decarboxylation reaction and the CDP-ethanolamine branch of the Kennedy pathway to PE formation in mammalian cells has been experimentally addressed using pathway-specific stable isotope labelling experiments, revealing a preferential use of the CDP-ethanolamine pathway over PS decar-boxylation in a ratio of approximately 2 : 1 [65] In addition, the two pathways were found to generate distinct PE molecular species, with the PS decarboxyl-ation route having a preference for long-chain, polyun-saturated molecular species Deletion of the ET gene

in mice causes embryonic lethality, indicating that PE levels cannot be maintained by PS decarboxylation [66]

In T brucei, de novo synthesis of PE occurs exclu-sively via the CDP-ethanolamine branch of the Ken-nedy pathway [36] All enzymes have been identified and experimentally validated [36,38,44] Disruption of the pathway by downregulation of any of the three

enzymes using RNAi results in growth arrest of the

parasites To our knowledge, T brucei represented

the first eukaryotic organism in which the PE branch

of the Kennedy pathway was shown to be essential

for cell growth Only very recently, the essential

nat-ure of the Kennedy pathway was also demonstrated

in Plasmodium berghei blood stage parasites [67]

Analysis of the phospholipid composition of T brucei

parasites after RNAi against ethanolamine kinase or

ET showed alterations not only in PE but also in PC

and PS levels [36,37] In addition, inhibition of PE

synthesis also blocked de novo synthesis of GPI

anchors and prevented ethanolamine phosphoglycerol

addition to eukaryotic elongation factor 1A [44],

demonstrating the above-mentioned

precursor–prod-uct relationship between PE and

ethanolamine-con-taining protein modifications Furthermore, ablation

of ET activity resulted in disruption of mitochondrial

morphology and ultrastructure [37], demonstrating

for the first time a direct effect of reduced PE levels

on mitochondrial integrity Interestingly, a similar

observation has recently been made in mitochondria

of mammalian cells Preliminary work showed that a

reduction in mitochondrial PE levels, after depletion

of PS decarboxylase capacity, caused alterations in

mitochondrial morphology and motility (J E Vance,

personal communication), suggesting that the effects

seen in T brucei may represent a widespread

phe-nomenon

Remarkably, T brucei PE consists of high levels of

ether-type molecular species [36,68,69] RNAi against

EPT and CEPT demonstrated that bulk alk-1-enyl-acyl

PE is synthesized by EPT, whereas diacyl-type PE is

primarily produced by CEPT [36] (Fig 1) It is

tempt-ing to speculate that the two enzymes may be involved

in generating two spatially and functionally distinct

pools of PE in T brucei

The contribution of phosphoethanolamine generated

via sphingosine-1-phosphate degradation to PE

forma-tion in T brucei has not been determined However, in

Leishmania parasites, this pathway was shown to be

essential if exogenous ethanolamine as the substrate

for the Kennedy pathway was absent from the culture

medium [70]

The pathway for PS formation in T brucei has not

been firmly established At present, it is unclear if PS

is synthesized from CDP-diacylglycerol and l-serine by

PS synthetase [38], or by head group exchange with

PE involving PS synthase-2 [37] Preliminary findings

in our laboratory using RNAi against a candidate gene

encoding PS synthase-2 indicate that PS formation is

essential for parasite viability (J Jelk & P Bu¨tikofer,

unpublished data)

Biosynthesis of phosphatidylglycerol (PG) and CL

PG and CL represent minor glycerophospholipid classes in eukaryotes CL is predominantly found in the inner mitochondrial membrane [52] or at contact sites

of inner and outer mitochondrial membranes [71] CL is rather unique in that it has a dimeric structure, consist-ing of two phosphatidyl moieties attached to glycerol and a small negatively charged head group, providing distinct physicochemical properties to CL and CL-containing membranes (reviewed in [72]) Among the many roles of CL, it has been shown to be required for proper function of key mitochondrial enzymes and proteins involved in ATP production via oxidative phosphorylation, as well as for mitochondrial transport systems (reviewed in [73–75]) In addition, CL organizes into membrane domains and participates in the formation and maintenance of dynamic protein–lipid and protein–protein interactions (reviewed in [76]) Remarkably, a reduction in CL levels and changes in the fatty acyl composition of CL have been linked to human diseases, such as Barth syndrome, an X-linked recessive human disorder caused by a defect in the enzyme tafazzin, which is involved in CL acyl chain remodelling in mammalian cells (reviewed in [77])

In contrast to their low abundance in eukaryotic cells, PG and CL represent the major anionic glycero-phospholipid classes in most positive and Gram-negative bacteria, accounting for 20 and 5% of total phospholipids, respectively [78] In both prokaryotes and eukaryotes, CL and its biosynthetic precursor,

PG, are synthesized from phosphatidic acid (reviewed

in [79]) Phosphatidic acid is first activated with CTP

to CDP-diacylglycerol by the enzyme CDP-diacylglyc-erol synthase Following condensation with glycCDP-diacylglyc-erol-3- glycerol-3-phosphate to phosphatidylglyceroglycerol-3-phosphate (PGP) by PGP synthase, the terminal phosphate group is hydro-lysed to form PG Interestingly, although bacterial enzymes catalysing PGP dephosphorylation were reported almost 30 years ago [80], the first eukaryotic PGP phosphatase has only recently been identified

in S cerevisiae [81] The final biosynthetic step in CL formation, catalysed by CL synthase, differs between prokaryotes and eukaryotes In prokaryotes, PG and a phosphatidyl moiety from another PG are condensed

to CL, whereas in eukaryotes, PG and CDP-diacyl-glycerol are fused to CL (reviewed in [79])

PGP synthase and CL synthase each belong to two distinct protein families The CDP-alcohol phosphat-idyltransferase family includes phosphatidyl- and phos-photransferases acting on CDP-alcohols, whereas the phospholipase D family contains phosphatidyltransferases

having active sites related to those found in

phospholi-pase D [57] Bacteria commonly use CDP-alcohol

phosphatidyltransferases for the PGP synthase

reac-tion, whereas mammals and yeast have phospholipase

D-like PGP synthases [23] Conversely, almost all

prokaryotes use phospholipase D-like enzymes for the

CL synthase reaction, whereas eukaryotic CL

synthas-es belong to the CDP-alcohol phosphatidyltransferase

family (reviewed in [23,79])

Experimental evidence for the presence of CL and

PG in both T brucei bloodstream and procyclic forms

has been reported [68,69] However, the pathway for

CL synthesis has not been elucidated Recently,

candi-date genes encoding enzymes for all steps in CL

syn-thesis have been identified using bioinformatic tools

[11] Preliminary results indicate that PG and CL

synthesis in T brucei is essential for parasite growth

(M Serricchio & P Bu¨tikofer, unpublished data)

Biosynthesis of phosphatidylinositol

(PI) and GPI

PI is a glycerophospholipid class containing an inositol

head group derived from the polyol, myo-inositol PI

or derivatives thereof are found in all eukaryotes,

including fungi and protozoa, but also in archaea and

some pathogenic bacteria [82,83] In eukaryotes, PI

constitutes 3–20% of cellular phospholipids [13] Apart

from being a structural membrane component, PI and

its phosphorylated forms also serve as precursors for

cell signalling molecules [84] and the biosynthesis of

GPI anchors (reviewed in [85,86])

Intracellular myo-inositol can be generated de novo in

a two-step reaction process involving

inositol-3-phos-phate synthase, generating inositol-3-phosinositol-3-phos-phate from

glucose-6-phosphate, and inositol monophosphatase,

catalysing dephosphorylation of inositol-3-phosphate to

inositol (reviewed in [84]) Alternatively, myo-inositol

can be taken up from the environment by inositol

trans-porters (reviewed in [87]) Subsequently, myo-inositol is

transferred to CDP-diacylglycerol by the action of PI

synthase, an enzyme that is conserved in all eukaryotes

[88]

In T brucei, the pathway for myo-inositol synthesis

and PI formation has been established It has been

proposed that T brucei bloodstream forms contain

two pools of PI synthase [89,90] One pool localizes to

the ER and uses myo-inositol generated de novo from

glucose-6-phosphate whereas the other pool associates

with the Golgi and uses myo-inositol taken up from

the environment (Fig 2) In T brucei bloodstream

forms, de novo formation of myo-inositol is essential

[90] In addition, recent results indicate that in

both procyclic- and bloodstream-form trypanosomes, uptake of myo-inositol via a specific transporter is nec-essary for normal growth (A Gonzalez Salgado &

P Bu¨tikofer, unpublished data) Interestingly, the PI pool formed from endogenously produced myo-inositol

is primarily used for GPI synthesis, whereas exogenous myo-inositol is used for bulk PI formation [89,90] This two-pool model is consistent with a previous report showing the presence of a subset of distinct PI molecu-lar species that is used for GPI anchor biosynthesis [91] However, it does not explain why cytosolic myo-inositol produced from glucose-6-phosphate does not (freely) exchange with myo-inositol taken up from the medium, unless de novo-synthesized myo-inositol or its precursor, myo-inositol-3-phosphate, are sequestered from exogenous myo-inositol, as has been suggested [90] PI synthase was found to be essential in both bloodstream- [89] and procyclic-form trypanosomes (M Serricchio & P Bu¨tikofer, unpublished data) Although several candidate genes encoding PI kinases have been identified in T brucei [11], the reactions leading to the production of phosphorylated PIs have not been examined experimentally In contrast, the involvement of PI as a precursor for GPI anchor synthe-sis has been extensively studied in both bloodstream-and procyclic-form T brucei (reviewed in [86]) In fact,

it was in T brucei where, for the first time, the entire GPI biosynthetic pathway leading to the formation of the GPI core precursor, ethanolamine-phosphate-Manal-2Manal-6Manal-GlcN-PI [92,93], and the first

Fig 2 Biosynthesis of inositol-containing lipids in T brucei In

T brucei bloodstream forms, two pools of PI synthase have been reported, one localizing to the ER and one to the Golgi [89] The corresponding model proposes that the ER enzyme preferentially uses inositol formed de novo from glucose-6-phosphate to generate PI for GPI anchor synthesis, whereas the Golgi enzyme primarily uses inositol taken up from the environment via a putative myo-inositol transporter (MIT) for PI and IPC synthesis It is not clear how the two pools of myo-inositol in the cytosol are seques-tered, or exchange with each other.

complete chemical structure of a GPI anchor [2], were

established In addition, T brucei was the first organism

in which remodelling of the acyl chain composition of

PI was demonstrated During GPI anchor synthesis and

after GPI attachment to protein in bloodstream-form

parasites, the PI acyl chains are replaced by myristate

[94,95] More recent data demonstrate that GPI lipid

remodelling also occurs in procyclic-form T congolense

and, possibly, T brucei [96] Following the discovery in

T brucei, remodelling of GPIs was also reported in

many other organisms (reviewed in [97,98]), indicating

that this process probably represents a general event

during GPI anchoring of proteins

Biosynthesis of sphingophospholipids

The sphingophospholipids, consisting of SM,

ethanol-aminephosphorylceramide (EPC) and

inositolphos-phorylceramide (IPC), represent key structural

components of virtually all eukaryotic membranes In

addition, they represent reservoirs for important

sig-nalling molecules, such as sphingosine,

sphingosine-1-phosphate and ceramide (reviewed in [99,100]) In most

mammalian cells, SM is by far the most abundant

sphingophospholipid class (reviewed in [13]), whereas

EPC, which represents the major sphingophospholipid

in Drosophila melanogaster [101], only occurs in trace

amounts [102] IPCs, or derivatives thereof, have not

been detected in mammalian cells, instead they

repre-sent prominent sphingophospholipid classes in fungi,

plants and protozoa [103–107]

The biosynthesis of sphingo(phospho)lipids starts

with the condensation of l-serine with palmitoyl-CoA

to form 3-ketosphinganine, a reaction catalysed by

serine palmitoyltransferase After reduction of the

product, dihydrosphinganine is N-acylated to

dihydro-ceramide by a family of dihydro-ceramide synthases, with its

members showing distinct substrate specificities for

fatty acyl-CoAs [108,109] Dihydroceramide is

subse-quently desaturated to ceramide, the central metabolite

in sphingolipid metabolism and branch point for the

synthesis of SM, EPC and IPC

The formation of SM is catalysed by SM synthase

and involves transfer of phosphocholine from PC to

ceramide to generate SM and diradylglycerol In

mam-malian cells, two SM synthases have been identified,

one located in the lumen of the Golgi and the other in

the plasma membrane [110] In addition, mammalian

cells express an SM synthase-related protein, SMSr,

that was recently shown to have EPC synthase activity

and is localized in the ER [111,112], confirming earlier

reports on the presence of such an activity in rat

liver and brain microsomes [102,113] The distinct

localization of the SM synthases presumably reflects their roles in de novo SM synthesis (Golgi enzyme) and regeneration of SM from ceramide (plasma membrane enzyme) IPC synthase is an essential enzyme in fungi [114,115] and localizes to the Golgi [116] Recently, its function and localization in yeast was shown to be dependent on the expression of an additional protein, Kei1, suggesting that IPC synthase may consist of multiple subunits [117]

In T brucei, candidate genes for all enzymes involved in ceramide synthesis have been identified [11] However, with the exception of serine palmitoyl-transferase [118,119], individual enzymes have not been studied experimentally In contrast, the subsequent steps in SM, IPC and EPC formation in T brucei have recently been characterized in detail [107,120] The reactions are catalysed by a family of sphingolipid syn-thases, TbSLS1-4, showing distinct substrate specifici-ties Using a cell-free synthesis system for the expression of polytopic membrane proteins [121], TbSLS1 was identified as IPC synthase, TbSLS2 as EPC synthase, whereas TbSLS3 and TbSLS4 show dual specificities for PC and PE as head group donors

to produce SM and EPC, respectively [120] Interest-ingly, the production of the different sphingophospho-lipid classes is developmentally regulated, with IPC being produced preferentially in T brucei procyclic forms and EPC in bloodstream forms, whereas SM is generated in both life cycle forms [107,120] (Fig 3) The localization of T brucei sphingolipid synthases has not been reported Whether sphingophospholipids are involved in protein trafficking to the cell surface in

T brucei bloodstream forms is unclear [35,119] To our knowledge, T brucei represents the only organism

so far that synthesizes all three sphingophospholipid classes, SM, IPC and EPC, and thus represents

an ideal model organism to study their biosynthesis, regulation and functional roles

Fig 3 Sphingophospholipid formation in T brucei In T brucei, all three classes of sphingophospholipids, EPC, IPC and SM, are gen-erated A family of sphingolipid synthases (TbSLS1–4) is responsi-ble for the stage-specific production of the different classes in bloodstream- (BSF) and procyclic-form (PCF) parasites The horizon-tal line indicates that the lipid class is present in trace amounts only.

Work in PB’s laboratory is supported by Swiss

National Science Foundation grant 31003A_130815

and Sinergia grant CRSII3_127300 We thank J.E

Vance for communicating unpublished findings, and I

Roditi and J.D Bangs for comments on parts of the

manuscript PB thanks S Harley and M Bu¨tikofer for

stimulation and input MS thanks K Durrer for

support and advice

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