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

REVIEW ARTICLE
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 brucei is 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:
Website: />(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.
FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS 1035
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 phosphocholine
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
Phospholipid biosynthesis in T. brucei M. Serricchio and P. Bu
¨
tikofer
1036 FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS
(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.
AB C

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.
M. Serricchio and P. Bu
¨
tikofer Phospholipid biosynthesis in T. brucei
FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS 1037
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 formation 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 sphingosine-1-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
Phospholipid biosynthesis in T. brucei M. Serricchio and P. Bu
¨
tikofer
1038 FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS

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 Gram-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 glycerol-3-

phosphate to phosphatidylglycerophosphate (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 fami ly contains phosphatidyltransferases
M. Serricchio and P. Bu
¨
tikofer Phospholipid biosynthesis in T. brucei
FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS 1039
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-phosphate 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.
Phospholipid biosynthesis in T. brucei M. Serricchio and P. Bu
¨

tikofer
1040 FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS
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 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.InT. 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.
M. Serricchio and P. Bu
¨
tikofer Phospholipid biosynthesis in T. brucei
FEBS Journal 278 (2011) 1035–1046 ª 2011 The Authors Journal compilation ª 2011 FEBS 1041
Acknowledgements
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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