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Biochimica et Biophysica Acta xx (2006) xxx – xxx
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Metabolic functions of glycosomes in trypanosomatids
Paul A.M. Michels a,⁎, Frédéric Bringaud b , Murielle Herman a , Véronique Hannaert a
a
b
Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry,
Université catholique de Louvain, ICP-TROP 74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium
Laboratoire de Génomique Fonctionnelle des Trypanosomatides, Université Victor Segalen Bordeaux 2, UMR-5162 CNRS,
146 rue Léo Saignat, 33076 Bordeaux cedex, France
Received 30 April 2006; received in revised form 17 August 2006; accepted 18 August 2006
Abstract
Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several
important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary
considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The
glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein
content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this
process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an
extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several
other processes such as the pentose-phosphate pathway, β-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines,
ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form
trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several
lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing
different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would,
through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Trypanosome; Glycosome; Glycolysis; Differentiation; Metabolic adaptation; Organelle turnover
1. Introduction
Glycosomes were initially discovered in Trypanosoma
brucei [1], the parasite that causes human African sleeping
sickness. These organelles appeared to contain most of the
glycolytic enzymes, hence their designation as glycosomes.
Compartmentation of glycolysis within organelles is unique; in
Abbreviations: ATG, autophagy-related gene; Gly3P, glycerol 3-phosphate;
DHAP, dihydroxyacetone phosphate; FH, fumarase; FRD, fumarate reductase;
GAT, glycosomal ABC transporter; GPO, glycerol-3-phosphate oxidase; HXK,
hexokinase; MCF, mitochondrial carrier family; PAT, peroxisomal ABC
transporter; PEP, phosphoenolpyruvate; PEPCK, PEP carboxykinase; PGK,
phosphoglycerate kinase; PFK, phosphofructokinase; PPDK, pyruvate phosphate dikinase; PTS, peroxisome-targeting signal; PYK, pyruvate kinase; TAO,
trypanosome alternative oxidase; TCA, tricarboxylic acid
⁎ Corresponding author. Tel.: +32 2 764 74 73; fax: +32 2 762 68 53.
E-mail address: [email protected] (P.A.M. Michels).
other organisms this metabolic process is essentially cytosolic.
Glycosomes were subsequently also found in related protozoa
(reviewed in [2–7]), all belonging to the Kinetoplastida, a group
that also comprises other parasites causing serious tropical
diseases in humans such as Trypanosoma cruzi and a variety of
Leishmania species, as well as parasites of other vertebrates,
invertebrates and plants, and free-living organisms. These
organelles belong to the peroxisome family, although initially
some doubt existed about this affiliation because of the absence
of catalase, the hallmark peroxisomal enzyme, from Trypanosoma and Leishmania glycosomes and the almost exclusively
glycolytic role of the organelles in T. brucei; glycolytic
enzymes comprise up to 90% of the glycosomal protein content
when these parasites live in the mammalian bloodstream. Yet,
like peroxisomes, these organelles are bounded by a single
phospholipid bilayer, contain no DNA and catalase could be
detected in glycosomes of some other kinetoplastids. Also
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enzymes of ether-lipid metabolism are often present in
glycosomes [8,9], although not in those of bloodstream-form
T. brucei. Furthermore, subsequent research showed that the
biogenesis of glycosomes occurs along similar routes as that of
peroxisomes, and involves homologous proteins (peroxins)
(reviewed in [7]), and more recent biochemical studies as well
as genomic and proteomic analyses revealed more similarities at
the metabolic level between glycosomes and other members of
the peroxisome family (see below, Sections 5, 6 and 7).
2. Glycosomes during the life-cycle of trypanosomatids
Many Kinetoplastida have a complicated life-cycle [10,11].
This holds particularly true for the best-studied kinetoplastid
organisms, the parasites belonging to the genera Trypanosoma
and Leishmania. These organisms are transmitted between their
mammalian hosts by insects. Both in the mammal and the
insect, they often transit through different parts of the body,
each time being exposed to highly different environmental
conditions. Most transitions involve the cellular differentiation
of the parasite enabling it to adapt to the new environment
encountered, or to prepare itself for the next one. For example,
T. brucei is introduced into the mammalian bloodstream by the
bite of a tsetse fly. There it finds a largely constant, glucose-rich
environment, allowing it to proliferate rapidly while producing
all its ATP by a high rate of aerobic glycolysis or, at places
where the oxygen level is low, by anaerobic glycolysis.
Sequestering of the major part of the glycolytic pathway inside
glycosomes has been shown essential for glycolysis, ATP
production and growth of these bloodstream-form trypanosomes [12–14], and provides them with a unique mechanism
for quickly switching from aerobic to anaerobic glycolysis (see
Section 3) [1,15,16]. This mechanism allows the trypanosomes
to overcome short periods of anaerobiosis, although it does not
enable them to synthesize sufficient ATP for sustaining growth
[17]. Mitochondrial systems such as the tricarboxylic acid
(TCA) cycle, respiratory chain and the oxidative phosphorylation machinery are largely repressed at this stage of the lifecycle (reviewed in [3,18–20]). Such glycolysis-dependent,
long-slender trypanosomes are not prepared for life in the insect
midgut, where the concentration of sugars is usually very low
and that of amino acids, notably proline and threonine high;
therefore, they quickly die when ingested by a next fly taking a
meal. However, in the bloodstream the long-slender forms may
differentiate into short-stumpy trypanosomes which are still
glycolysis-dependent but have a partially de-repressed mitochondrial system, allowing them to survive in the insect midgut.
Upon ingestion, these short-stumpy trypanosomes differentiate
rapidly into so-called procyclic forms with a much more
elaborate mitochondrial system, and also a considerably
different, more extensive glycosomal metabolic network (see
Sections 4 and 5). In a period of 2–3 weeks, the trypanosomes
move from the midgut, via the foregut and proboscis to the
salivary glands, while undergoing additional differentiation
steps resulting in morphologically distinct forms [21] which so
far have not yet been metabolically characterized. It is likely
that the metabolic repertoire of the glycosomes varies with the
different environments encountered by each of these differentiated forms. The mammalian-infective metacyclic form, that
finally develops in the salivary glands, is prepared again for an
exclusively glycolytic metabolism in the mammalian blood.
In contrast to T. brucei, which resides extracellularly
throughout its life-cycle, T. cruzi and Leishmania species
have additional, intracellular stages in their mammalian hosts:
T. cruzi in the cytosol of various tissues, Leishmania in the
lysosomes of macrophages. Consequently, their metabolism has
to be able to rely also on the nutrients available in these highly
specific environments, with consequences for the adaptability
of their metabolism. Moreover, these parasites are transmitted
by different insects (triatomine bugs and sand flies, respectively) and follow also different tracks within the insect vector. A
need for metabolic adaptability to cope with different environments is possibly also important for each of the many other,
biochemically less well-characterized kinetoplastids irrespective whether they infect one or more specific hosts or are free
living. As will be argued below, glycosomes are likely to play
an important role in the metabolic adaptation to each of the
environments to which kinetoplastid organisms are exposed.
3. Compartmentation of glycolysis in glycosomes in
bloodstream-form T. brucei
When parasitizing the mammalian bloodstream, the African
trypanosome T. brucei is, for its free-energy needs, totally
dependent on a continuous supply of glucose present in the
blood and body fluids of its host. All ATP synthesized by this
organism comes from the conversion of glucose into pyruvate,
the end-product of trypanosome glycolysis in this vertebrate
stage. The seven enzymes involved in the conversion of glucose
into 3-phosphoglycerate are present inside the glycosome, while
those catalyzing the last part of the pathway are localized in the
cytosol (Fig. 1) [1]. The pyruvate is excreted into the host's
bloodstream. Within the glycosome, consumption and production of ATP and NADH by glycolysis are balanced: the
intraglycosomal consumption of ATP by hexokinase (HXK)
and phosphofructokinase (PFK) is compensated by the ATP
production by phosphoglycerate kinase (PGK). Net ATP
formation occurs in the cytosol, in the reaction catalyzed by
pyruvate kinase (PYK). Similarly, the glycosomal NADH
resulting from the reaction catalyzed by glyceraldehyde-3phosphate dehydrogenase is re-oxidized inside the organelle
followed by the transfer of the electrons to O2 via a
mitochondrial glycerol-3-phosphate oxidase (GPO). This process involves a glycosomal NADH-dependent glycerol-3phosphate dehydrogenase, a putative transporter (‘shuttle’) in
the glycosomal membrane which exchanges glycerol 3phosphate (Gly3P) for dihydroxyacetone phosphate (DHAP),
and the mitochondrial GPO. The GPO is in fact a system
comprising an FAD-linked glycerol-3-phosphate dehydrogenase that is most likely associated with the outer surface of the
mitochondrial inner membrane, ubiquinone and a terminal
oxidase, known as the trypanosome alternative oxidase (TAO).
This respiratory process seems not to be involved in free-energy
transduction. The TAO is insensitive to cyanide, but can be
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3
Fig. 1. Schematic representation of glycolysis in the bloodstream-form of T. brucei. Under aerobic conditions, glucose is converted into pyruvate. Under anaerobic
conditions equimolar amounts of glycerol and pyruvate are produced. Abbreviations: 1,3BPGA, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; F-6-P,
fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; G-3-P, glyceraldehyde 3-phosphate; G-6-P, glucose 6-phosphate; Gly-3-P, glycerol 3-phosphate; PEP,
phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Pi, inorganic phosphate; UQ, ubiquinone pool. Enzymes are: 1, hexokinase: 2, glucose-6-phosphate isomerase; 3,
phosphofructokinase; 4, aldolase; 5, triosephosphate isomerase; 6, glycerol-3-phosphate dehydrogenase; 7, glycerol kinase; 8, glyceraldehyde-3-phosphate
dehydrogenase; 9, glycosomal phosphoglycerate kinase; 10, phosphoglycerate mutase; 11, enolase; 12, pyruvate kinase; 13, FAD-dependent glycerol-3-phosphate
dehydrogenase; 14, alternative oxidase.
inhibited by salicylhydroxamic acid. Inhibition of TAO mimics
the effect of a lack of oxygen on carbohydrate metabolism of the
bloodstream form. Under these conditions, NAD+ is regenerated
by the reduction of DHAP to Gly3P, followed by the formation
of glycerol that is excreted by the organism. This is achieved by
reversal of the glycerol kinase reaction, thus synthesizing
glycerol and ATP from Gly3P and ADP [1,15,16]. However, this
reverse reaction is thermodynamically unfavourable and only
feasible by mass action at high Gly3P concentration and a high
ADP/ATP ratio, conditions that are difficult to attain in an entire
cell but may occur in a small, closed compartment. By this
reverse reaction, the ATP/ADP balance within the glycosomes is
maintained; the ATP formed in the glycerol kinase reaction
compensates for the loss of one molecule of ATP in the PGK
reaction, because now one molecule of 3-phosphoglycerate,
rather than two, is produced per molecule of glucose consumed.
As a consequence, glucose is dismutated into equimolar
amounts of pyruvate and glycerol, with net synthesis of only
one molecule of ATP.
The regulation of glycolysis in trypanosomes differs largely
from that of other organisms. There is an apparent lack of
activity regulation of the glycolytic enzymes HXK and PFK
[22–24]. In most organisms, the activities of these two key
enzymes are highly regulated by their products, by metabolites
further downstream in the metabolism or by other (allosteric)
effectors [25]. This regulation serves two purposes. First, it
prevents the loss of ATP by futile cycling when glycolysis and
gluconeogenesis occur simultaneously. Second, it has been
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argued, and experimentally confirmed by using Saccharomyces
cerevisiae mutants, that the ‘turbo design’ of glycolysis requires
a tight regulation of the first steps of glycolysis [26]. Indeed,
absence of regulation may lead to unrestricted accumulation of
glycolytic intermediates, a situation that will be highly toxic for
the cell.
Proper compartmentation of glycolytic enzymes inside
glycosomes and possession of intact organelles are essential
for the survival of bloodstream-form T. brucei. Indeed, several
experiments have provided evidence that mislocalization of
glycolytic enzymes is deleterious for the parasite. Trypanosomes contain two major isoenzymes of PGK located in
different compartments of the cell: one (PGKg), present in
glycosomes, is only expressed in the mammalian bloodstreamform parasites, while a cytosolic enzyme (PGKc) operates
predominantly in procyclic trypanosomes [27,28]. Bloodstream-form trypanosomes were rapidly killed when, from an
additional ectopic gene, PGKc or a truncated form of PGKg
without the glycosome-targeting signal (PTS1) was expressed
[29]. Expression of an inactive PGKc had no effect, indicating
that the toxicity depends both on enzyme activity and cytosolic
location. Similar growth inhibition has been observed after
transfection of bloodstream-form T. brucei with the gene of S.
cerevisiae triosephosphate isomerase expressed as a cytosolic
enzyme [17]. Furthermore, partial relocation of glycosomal
matrix proteins to the cytosol upon depletion of peroxins PEX2,
6, 10, 12 or 14 by RNA interference (RNAi) readily killed
bloodstream-form cells [13,14,30,31].
To understand why the presence of a glycolytic enzyme in
the wrong compartment is detrimental for trypanosomes, one
has to know the function or consequences of this form of
metabolic compartmentation. To this aim, a mathematical
model of glycolysis in bloodstream-form T. brucei was
developed on the basis of kinetic data as determined for the
different glycolytic enzymes [32]. One question addressed was
how the functional behaviour of trypanosome glycolysis would
change if the pathway were not compartmentalized [12].
According to the computer model this would not significantly
affect the steady-state glycolytic flux. But, strikingly, it will
lead to toxic accumulation of hexose-phosphates upon addition
of glucose. Such toxic accumulation is prevented by
compartmentation, since the kinases at the beginning of the
pathway respond to the glycosomal ATP/ADP ratio, not the
cytosolic one. The glycosomal ATP/ADP ratio, which,
according to the computer model, is usually low, controls the
activity of HXK and PFK and maintains the concentration of
the hexose-phosphates constrained within a narrow range. If
the enzymes would sense the higher cytosolic ATP/ADP ratio,
their activity would not be restrained and the intermediates
would accumulate. Therefore, the computer analysis suggested
that the sequestering of the glycolytic pathway within a
membrane that is poorly permeable for solutes is essential for
the parasite, because it compensates for the lack of activity
regulation of its enzymes. Indeed, experimental data have been
obtained to support the notion that compartmentation of
glycolysis serves such a regulatory function and will be
presented in Section 4.
4. Glycosomes and energy metabolism in insect-stage
T. brucei and other trypanosomatids
During its journey through the tsetse fly, T. brucei undergoes
several differentiation steps, adapting itself to each new
environment encountered. The procyclic form living in the
midgut is, with the bloodstream form, the only one that so far
could be axenically cultured and has thus been made amenable
to biochemical investigations. Also the promastigote Leishmania spp. and epimastigote T. cruzi, which propagate in the
midgut of their respective insect vectors, are commonly studied
in laboratories. Although the metabolic networks, both within
the glycosomes and the fully developed mitochondrion, of all
these insect-stage trypanosomatids seem very similar, most of
the recent data regarding glycosomes and energy metabolism
pertain to procyclic trypanosomes, since gene silencing by
RNAi could be extensively used in T. brucei [33,34], but failed
to be functional in the other parasites [35,36].
When grown in commonly used glucose-rich media,
procyclic T. brucei and epimastigote T. cruzi primarily use the
glucose as carbon source [37,38], whereas amino acids
(including L-proline) are the main energy source present in
their insect vector [39]. In glucose-depleted medium, however,
procyclic trypanosomes considerably increase the rate of Lproline consumption (6-fold), indicating that it is the major
carbon source used by the parasite in its natural environment
and glucose, when present, exerts a negative control on Lproline metabolism [40]. Glucose is catabolized in the
glycosomes, as described above for the bloodstream-form
trypanosome (see Section 3), whereas L-proline degradation
takes place in the mitochondrion. Glycolysis appeared dispensable, since procyclics of several strains have been successfully
adapted to glucose-depleted media with no significant effect on
growth rate [40,41]. Interesting is the non-essential role of
glycosomes in procyclic trypanosomes grown in glucosedepleted media, as exemplified by the analysis of a PEX14
knock-down mutant [14]. Procyclic trypanosomes were killed
by RNAi-dependent depletion of PEX14 only when glucose
was present, suggesting that glycosomes are only essential to
compartmentalize a functional glycolytic pathway. This observation strengthens the hypothesis that glycosomes serve to
prevent the dangerous effect of the turbo design of glycolysis
[12,26] (see Section 3). Experimental support for this
hypothesis was provided by the observation that RNAimediated knockdown of HXK rescued procyclic cells from
glucose toxicity, even though glycosomal proteins were
mislocalized to the cytosol [42].
The extensive tracheal system of insects renders all organs
and tissues, including the digestive tractus, highly aerobic [43].
Consequently, insect parasites have developed an aerobic
metabolism, exemplified by the presence of two distinct mitochondrial terminal oxidases (the cyanide-sensitive cytochrome
oxidase and the salicylhydroxamic acid-sensitive TAO), both
using oxygen as electron sink. Indeed, the midgut trypanosomatids cannot adapt to long-term growth under anaerobic
conditions and go into reversible or irreversible metabolic arrest
during anoxia [44–46]. Although they are dependent on oxygen
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for proliferation, the insect-stage trypanosomatids developed a
fermentative-like metabolism, since glucose (and L-proline) is
converted into partially oxidized end-products (succinate,
acetate, lactate, ethanol and/or glycerol, depending on the
species) by so-called aerobic fermentation [47].
In these insect-stage trypanosomes too, glucose is essentially
metabolized inside the glycosomes (Fig. 2), but with three main
differences compared with the bloodstream form of the parasite
(for recent reviews see [5,18–20]). First, PGK is present in the
cytosol and, therefore, 3-phosphoglycerate is produced in this
compartment [27]. Second, phosphoenolpyruvate (PEP) produced in the cytosol forms a metabolic branching point and is
the substrate for two glycosomal kinases, i.e., PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK)
5
[48–50]. The PEP-CO2 condensation catalyzed by PEPCK is
the initial step to the production, inside glycosomes, of
succinate, a major end-product excreted by most trypanosomatids [20,46,47,51,52]. Third, instead of being the final endproduct, pyruvate is further converted into acetate, lactate and Lalanine in the mitochondrion and/or the cytosol [46,51,53,54].
The two former changes imply that the mechanism by with the
redox potential and ATP/ADP ratio are balanced within
glycosomes of procyclic trypanosomes differs from that in the
bloodstream form of T. brucei.
The production of succinate from PEP involves two
glycosomal NADH-dependent oxido-reductases, malate dehydrogenase and fumarate reductase (FRDg) [49,51], which
provide a means for the re-oxidation of NADH produced in
Fig. 2. Schematic representation of glucose metabolism in the procyclic form of T. brucei. Excreted end products of glucose metabolism (acetate, L-alanine, glycerol,
L-lactate, succinate and CO2) are in white characters on a black background. Arrows with different thicknesses tentatively represent the metabolic flux at each
enzymatic step. Dashed arrows indicate steps which are supposed to occur at a background level or not at all. The mitochondrial outer membrane is permeable to
metabolites and is only shown in the vicinity of the schematic electron-transport chain. Abbreviations: AA, amino acid; 1,3BPGA, 1,3-bisphosphoglycerate; C,
cytochrome c; CoASH, coenzyme A; DHAP, dihydroxyacetone phosphate; F-6-P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; G-3-P, glyceraldehyde 3phosphate; G-6-P, glucose 6-phosphate; Gly-3-P, glycerol 3-phosphate; OA, 2-oxoacid; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Pi, inorganic
phosphate; PPi, inorganic pyrophosphate; SucCoA, succinyl-CoA; UQ, ubiquinone pool. Enzymes are: 1, hexokinase: 2, glucose-6-phosphate isomerase; 3,
phosphofructokinase; 4, aldolase; 5, triosephosphate isomerase; 6, glycerol-3-phosphate dehydrogenase; 7, glycerol kinase; 8, glyceraldehyde-3-phosphate
dehydrogenase; 9, glycosomal phosphoglycerate kinase; 10, cytosolic phosphoglycerate kinase; 11, phosphoglycerate mutase; 12, enolase; 13, pyruvate kinase; 14,
phosphoenolpyruvate carboxykinase; 15, pyruvate phosphate dikinase; 16, glycosomal malate dehydrogenase; 17, cytosolic (and glycosomal) fumarase (FHc); 18,
glycosomal NADH-dependent fumarate reductase; 19, mitochondrial fumarase (FHm); 20, mitochondrial NADH-dependent fumarate reductase; 21, glycosomal
adenylate kinase; 22, malic enzyme; 23, unknown enzyme; 24, alanine aminotransferase; 25, pyruvate dehydrogenase complex; 26, acetate:succinate CoA-transferase;
27, unknown enzyme; 28, succinyl-CoA synthetase; 29, FAD-dependent glycerol-3-phosphate dehydrogenase; 30, rotenone-insensitive NADH dehydrogenase; 31,
alternative oxidase; 32, F0F1-ATP synthase; I, II, III and IV, complexes of the respiratory chain.
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the organelle by the glycolytic glyceraldehyde-3-phosphate
dehydrogenase. Theoretically, two NADH molecules are
consumed per molecule of succinate produced for a single
NADH molecule produced per molecule of PEP synthesized
from glucose, implying that only half of PEP produced in the
cytosol needs to be re-routed to the organelle and converted into
succinate in order to balance the NADH/NAD+ ratio. However,
the fraction of PEP metabolized in the glycosome is
significantly higher than expected, since approximately 70%
of the glucose consumed is converted into succinate [51,55].
This apparent discrepancy probably results from the existence
of a second succinate production pathway, located in the
mitochondrion, fed by malate produced in the glycosomes (Fig.
2), which accounts for 14–44% of succinate produced from the
glucose metabolism [52]. For each succinate molecule produced
in the mitochondrion, a single NADH molecule is formed in the
glycosomes, instead of two. Assuming that the glycosomal
succinic fermentation is sufficient for maintaining the glycosomal redox balance, the Gly3P/DHAP shuttle described for the
bloodstream-form T. brucei seems a superfluous pathway in the
procyclic cells. However, also this insect form of the parasite
expresses a functional shuttle, providing additional metabolic
flexibility that may be beneficial under particular growth
conditions [56]. This is exemplified by the observation that
RNAi-dependent depletion of the glycosomal FRD activity
does not affect growth rate while glucose is still consumed,
albeit at a reduced level, suggesting that an alternative way
(possibly the Gly3P/DHAP shuttle) is used by this mutant to
substitute for NADH oxidation by FRDg [51].
Surprisingly, the glycosomal succinic fermentation pathway
may make a detour to the cytosolic compartment (Fig. 2). Two
class I fumarases, respectively located in the cytosol (FHc) and
the mitochondrion (FHm), have recently been identified,
suggesting the absence of fumarase in the glycosomes [57].
Indeed, a recent proteomic analysis of glycosomes isolated
from procyclic trypanosomes did not reveal a fumarase [58],
but contrasts with the previous detection of significant FH
activity in glycosomal fractions of procyclic trypanosomes
[51]. These contradictory data may be correlated to the
presence of a cryptic PTS1 at the C-terminal extremity of T.
brucei FHc (AKLV). One could imagine that this cryptic PTS1
becomes functional after a form of decrypting (by the activity
of a carboxypeptidase) dependent on environmental conditions. A similar cryptic PTS1 is present at the C-terminal
extremity of the orthologous T. cruzi FH (SKLL), whereas a
somewhat different sequence is found in the L. major enzyme
(SKTLA). In the latter case, but also for the Trypanosoma
species, alternative possibilities to explain the dual localization
are the existence of non-consensus targeting sequences or
piggy-back transport as discussed in more detail elsewhere [7].
Nonetheless, whatever the import signal and mechanism, the
variability in the dual localization suggests a regulation of the
import process. The unexpected cytosolic location of FHc may
be attributed to the requirement of fumarate (instead of NAD+)
as electron acceptor for the cytosolic dihydroorotate dehydrogenase, an essential enzyme of de novo pyrimidine biosynthesis [59,60].
The relocation of PGK to the cytosol of trypanosomes when
they differentiate from bloodstream to procyclic forms results
from the expression of a different isoenzyme, PGKc instead of
PGKg. This change has been interpreted in relation to the
necessity to balance the ATP production and consumption
inside the glycosomes: in procyclics the phosphorylation of the
glycolytically produced ADP would be performed by PEPCK
during the glycosomal succinic fermentation. However, the
conversion of a significant part of PEP into pyruvate by the
cytosolic PYK implies that the PEPCK reaction would not
contribute sufficiently to maintaining the glycosomal ATP/ADP
balance. Consequently, at least another glycosomal ATPproducing step is required to complete the metabolic scheme.
This function may be fulfilled by a constitutively expressed
third PGK isoform, the glycosomal PGKA [27,28,61,62]. The
dual localization of PGK activity (PGKA and PGKc) may help
to maintain the ATP balance inside the glycosomes of procyclic
trypanosomes by altering the ratio of the fluxes from 1,3bisphosphoglycerate to 3-phosphoglycerate via the two different routes (Fig. 2). However, the contribution by PGKA to
glycosomal ATP production is debated, since its relative amount
is very low compared to the other isoforms in T. brucei, it has a
low specific activity, and its activity is not detectable in
glycosomal fractions of procyclic trypanosomes ([48], and FB,
unpublished data). The alternative of glycosomal ATP production by glycerol kinase is thermodynamically unfavoured, and
thus unlikely, and indeed the procyclic form does not excrete
significant amounts of glycerol when grown under standard
conditions [46,47,51]. PPDK is another glycosomal ATPproducing enzyme, which utilizes PEP, AMP and PPi to
produce pyruvate, ATP and Pi [50,63]. This PPi-dependent
enzyme was proposed to substitute for pyrophosphatase in order
to hydrolyze the PPi produced in the glycosomes by
biosynthetic pathways (see Section 5) which would otherwise
be inhibited [4,5,50,63]. This hypothesis implies that the rate of
ATP production by PPDK depends on the flux of the
glycosomal PPi-generating pathways, which should be significantly lower than the catabolic flux of glucose. Consequently,
the PPDK activity is probably not sufficient to maintain the
glycosomal ATP/ADP balance, as confirmed experimentally,
since the viability and glucose metabolism are not affected by
PPDK gene knockout [38]. Alternatively, ATP could be
imported from outside the glycosomes, similar as reported for
peroxisomes, where the yeast Ant1p and human PMP34
nucleotide transporters are involved in the exchange of AMPADP and ATP across the membrane, to provide intraperoxisomal ATP for β-oxidation or activation of fatty acids,
respectively [64,65]. Indeed, the recent proteomic analysis of
glycosomes from procyclic trypanosomes identified a protein
homologous to mitochondrial ATP/ADP carriers, but a
mitochondrial contamination of the glycosomal-enriched fraction cannot be excluded [58]. The presence of several ATPproducing pathways in addition to a putative nucleotide
transporter may provide an important metabolic flexibility for
adaptation to rapid environmental changes. However, questions
remain as to how the glycosomal ATP/ADP balance is
maintained in procyclic trypanosomatids.
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Adaptive forms of all trypanosomatids analyzed to date,
except the slender bloodstream form of T. brucei, express the
NADH-dependent FRD activity [51,66–69] and produce
significant amounts of excreted succinate from glucose
[20,47]. Interestingly, succinate production seems to be
correlated with the presence of a PGK activity in the cytosol,
since the bloodstream form of T. brucei is the only one lacking
the PGKc isoform. All other trypanosomatid forms analyzed
express at least one cytosolic PGK isoform, often simultaneously with glycosomal isoforms [27,28,70–72]. In other
words, the exclusively glycosomal localization of PGK is
restricted to bloodstream-form T. brucei and correlates with the
absence of the glycosomal succinic fermentation pathway.
Thus, the repression of the succinic fermentation pathway by
bloodstream-form T. brucei is accompanied with the repression
of the cytosolic PGK in favour of the glycosomal isoform in
order to balance the glycosomal ATP/ADP ratio. These
adaptations to the glucose-rich environment of the vertebrate
blood came along with the repression of the mitochondrial TCA
cycle enzymes and respiratory chain. Interestingly this
expression strategy appears to be a recent evolutionary
adaptation by T. brucei, because it is not found in all other
trypanosomatids including its closest known relative, T.
congolense, which has a very similar life cycle; it also
propagates in the blood of vertebrates after transmission by
tsetse flies. The bloodstream form of T. congolense excretes
succinate and expresses both the glycosomal and cytosolic PGK
isoforms [47,70]. The T. congolense orthologous gene (c2PGK)
of the T. brucei glycosomal PGKg gene encodes a cytosolic
isoform. Consequently, PGKA is the only glycosomal isoform
expressed in T. congolense [70]. In conclusion, all trypanosomatids analyzed so far developed the largely glycosomallylocated metabolic network for aerobic fermentation of glucose.
Only the long-slender bloodstream form of T. brucei could
afford to considerably reduce this network to the mere
glycolytic pathway, possibly as a result of its constant and
glucose-rich environment.
5. Other metabolic systems in glycosomes
Besides the glycolytic pathway and its extra branches
towards glycerol and succinate production, many other
enzymes, often parts of pathways, have been found inside
glycosomes during the last 20 years (Fig. 3). Most of these
enzymes were not, or only at very low activity, detectable in
bloodstream-form T. brucei, but were shown to be present in
glycosomes of cultured procyclic T. brucei and/or insect- or
mammalian-stage cells of Leishmania species or T. cruzi,
mostly upon cell fractionation, sometimes by immunofluorescence studies with intact cells. And indeed most of the enzymes
were shown to possess a PTS1 or PTS2. For example, the key
enzyme of the gluconeogenic pathway, fructose-1,6-bisphosphatase, possesses peroxisome-targeting signals [73] and
indeed was demonstrated to be present in glycosomes of T.
brucei. The observation that this enzyme is expressed
simultaneously with the glycolytic PFK (VH, unpublished
result), and the apparent absence from trypanosomatid PFK of
7
activity regulation mechanisms found in other organisms
[23,24] raise the still unsolved question as to how futile ATP
hydrolysis resulting from the combined activity of these two
glycosomal enzymes is avoided.
Also another glucose consuming process, the oxidative
branch of the pentose-phosphate pathway, is compartmentalized
inside glycosomes [74,75]. Although most of the enzymes of
this pathway in the three trypanosomatids for which the genome
sequencing has been completed appear to have a PTS, often a
substantial part of their activity was found in the cytosol,
indicating only partial compartmentation; in T. cruzi the
cytosolic contribution is even predominant [76]. Intriguingly,
genes encoding a sedoheptulose-1,7-bisphosphatase with a
PTS1 have been found in T. brucei and T. cruzi. This is a Calvin
cycle enzyme, usually only found in plastids of plants and algae
and in some fungi and ciliates where it is possibly present in the
cytosol exerting a still unknown function. Possibly the
trypanosomatids acquired the sedoheptulose-1,7-bisphosphatase by lateral gene transfer [73,77]; we hypothesize that the
parasites employ it in a modified pentose-phosphate pathway
(Fig. 3A), as described in more detail elsewhere [5].
All trypanosomatids studied contain various isoforms of
adenylate kinase, an enzyme that is important for ATP
homeostasis. In T. brucei seven genes encoding the enzyme
for several cell compartments have been found, including one
for a glycosomal enzyme [78]. The importance of this enzyme
for glycosomal metabolism has been shown by computer
simulation of metabolism and expression knockdown using
RNAi [32,78].
A glycosomal localization has previously also been reported
for most enzymes of the purine salvage pathway in different
trypanosomatids and for a bifunctional enzyme, resulting from a
gene fusion, catalyzing the last two of the six steps usually
comprising the de novo pyrimidine biosynthetic pathway,
whereas the other four enzymes were found in the cytosol
(Fig. 3B and C) (reviewed in [4,79]). Interestingly, the genes of
the enzymes for pyrimidine biosynthesis are organized in a
highly unusual way for trypanosomatids, i.e., in an operon-like
manner, similar to that in cyanobacteria and not found
elsewhere [80]. This organization and the sequence similarity
strongly suggest that these genes were acquired by lateral gene
transfer. As mammalian peroxisomes, trypanosomatid glycosomes have been shown to contain the first two steps of etherlipid biosynthesis, alkyl-DHAP synthase and DHAP acyltransferase (Fig. 3F) [8,9]. Also common with peroxisomes is the
association with glycosomes of some enzymes of β-oxidation
of fatty acids, such as 2-enoyl-CoA hydratase and an NADPHdependent 3-hydroxyacyl-CoA dehydrogenase (Fig. 3E)
[81,82]. For T. brucei it was shown that these two enzymes
behave as a bifunctional enzyme [82].
As mentioned above, the peroxisomal marker enzyme
catalase is absent from Trypanosoma and Leishmania species,
but it has been found in other kinetoplastids such as Crithidia
luciliae, Phytomonas sp. and Trypanoplasma borelli, where it
was located in the glycosomes [2,3]. But in recent years, a
different peroxide detoxification system has been unravelled
in trypanosomatids and components of it were found also in
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the glycosomes, in line with the possession of a PTS by
some of them [83]. The unique trypanosomatid system is a
cascade comprising a modified form of glutathione, N1,N8-bis
(glutathionyl)spermidine designated trypanothione, trypanothione reductase, a thioredoxin-related protein called tryparedoxin and 2-Cys-peroxiredoxin-type tryparedoxin
peroxidases and glutathione peroxidases (Fig. 3H) (reviewed
in [84]). Also other enzymes involved in oxidative stress
defence have been detected in glycosomes such as one of the
four iron-dependent superoxide dismutase isoenzymes of trypanosomatids [85,86].
The recently completed genome sequencing projects for T.
brucei, T. cruzi and L. major [87] have allowed to get further
insight into the metabolic repertoire of glycosomes by searching
all protein sequences with either a potential C-terminal PTS1 or
an N-terminal PTS2 [80]. Moreover, proteomic analyses of
glycosomes purified from both bloodstream- and procyclicform T. brucei have been initiated [58]. In the bioinformatic
analysis of L. major, 191 potential PTS1-containing proteins
and 68 potential PTS2-containing proteins with homologues in
T. brucei and T. cruzi were identified. About 50% of these were
hypothetical proteins to which no function could be attributed as
Fig. 3. Overview of glycosomal metabolic processes other than glycolysis. Pathways or single reactions are depicted of which enzymes are probably at least in part
sequestered in glycosomes of some life-cycle stages of T. brucei, T. cruzi and/or Leishmania species, as concluded from experiments involving cell fractionation in
conjunction with activity assays, immunoblotting and/or mass-spectrometry based proteomics, or immunofluorescence microscopy of intact cells and/or from the
identification of a (putative) peroxisome-targeting signal in the amino-acid sequence. The two circles given at each reaction indicate the probability of a glycosomal
localization of the enzyme: the left circle signifies the availability of experimental support (filled circle) or the lack (so far) of such support (open circle); a filled right
circle indicates the presence of a (potential) PTS, an open circle no detectable PTS. The pathways or processes indicated are: (A) pentose-phosphate pathway, (B)
purine salvage, (C) pyrimidine biosynthesis, (D) energy homeostasis, (E) β-oxidation, (F) ether-lipid biosynthesis, (G) squalene synthesis, (H) oxidative-stress
protection. In addition to the enzymes highlighted here many more candidate glycosomal enzymes and hypothetical proteins have been identified by genomic searches
and proteomic analysis [80,58]. The sedoheptulose-1,7-bisphosphatase is proposed to connect the glycolytic and pentose-phosphate pathways, allowing to vary the
yield for NADH and ATP from glycose metabolism according to the cells requirements, as discussed in more detail in [5]. For a more extensive discussion of the
various reactions, the subcellular localization of the enzymes and their expression in the different trypanosomatids life-cycle stages, see the main text and references
[4,5,58,80].
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9
Fig. 3 (continued ).
yet. The results of these genomic and proteomic studies largely
confirm the earlier experimental findings described above,
while filling in some gaps in pathways (Fig. 3). However, the
proteomic study did not detect any enzymes of β-oxidation,
although two enzymes were found with a candidate PTS2 in all
three trypanosomatids: the bifunctional 2-enoyl-CoA hydratase/
3-hydroxyacyl-CoA dehydrogenase mentioned above and 3ketoacyl-CoA thiolase. Possibly, the proteomic analysis was
performed with trypanosomes cultured under conditions by
which the pathway was repressed. However, the bifunctional
enzyme may also be targeted differently, since its sequence
contains also a candidate mitochondrial transit peptide.
Additionally, the genome sequences of T. cruzi and L. major
revealed candidate glycosomal enzymes that should allow these
parasites to feed the glycolytic pathway also with various other
sugars than glucose [80].
The glycosome also harbours some enzymes of the
mevalonate pathway for the biosynthesis of isoprenoids,
precursors of many important compounds such as squalene
and eventually sterols (Fig. 3G). Three enzymes, responsible for
the conversion of mevalonate to 5-pyrophospho-mevalonate
were each shown to possess a PTS in at least two of the three
trypanosomatids. The same holds true for a fourth enzyme
further in the pathway, squalene synthase, converting farnesylpyrophosphate into squalene. The first enzyme of the pathway,
3-hydroxy-3-methyl-glutaryl-CoA reductase had previously
been localized to mitochondrion and glycosomes [88]; its
sequence has indeed a candidate mitochondrial transit peptide
but not an identifiable PTS. The proteomic analysis detected the
mevalonate kinase only in glycosomes of procyclic cells.
In addition to adenylate kinase mentioned above, glycosomes may contain another enzyme involved in ATP homeostasis, arginine kinase (Fig. 3H). T. brucei and T. cruzi, but not
Leishmania species, contain this enzyme; in T. brucei even
three genes were identified coding for enzymes differing only in
their N- and C-termini. One of them has a putative PTS1.
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Indeed, the proteomic analysis identified the enzyme in
glycosomes of bloodstream- but not procyclic-form trypanosomes. Studies with T. cruzi have shown that phospho-arginine
is particularly important during periods of stress; it is likely that
this phosphagen acts as an energy reserve during differentiation
steps [89,90].
6. Metabolite transporters in the glycosomal membrane
As for peroxisomes, several lines of evidence provide strong
support for the notion that the membrane of glycosomes is
poorly permeable (reviewed in [7]). First, the biphasic kinetics
of radioactive labelling of glycolytic intermediates, when
trypanosomes received a pulse of 14C-glucose, suggested the
existence of two pools of intermediates: a rapidly labelled
glycosomal one, representing 20–30% of the total pool of
glycolytic metabolites and a slowly labelled cytosolic pool
representing 70–80% of the intermediates [91]. Exchange
between both pools of intermediates appeared to happen only
very slowly. Second, under anaerobic conditions bloodstreamform trypanosomes are still capable of performing glycolysis by
producing one molecule of pyruvate and one molecule of
glycerol per molecule of glucose consumed instead of two
molecules of pyruvate. As discussed in Section 3, this involves
the reversal of the glycerol kinase reaction, driven by
accumulation of Gly3P and a low ATP/ADP ratio as can only
be achieved in a small, closed compartment separate from the
cytosol. Further strong indications for the glycosomal membrane as a permeability barrier are the above mentioned
importance of maintaining a redox (NADH/NAD+) balance,
the apparent requirement of stoichiometry for the sum of
reactions inside the organelle [32], and the observed detrimental
effects resulting from mislocalization of glycolytic enzymes
[17,29].
The existence of the permeability barrier implies the need for
transporter molecules or pores for the exchange of specific
substrates, products and intermediates. So far, two groups of
candidate solute carriers, homologous to known transporters of
peroxisomal membranes [64,92] (see also elsewhere in this
issue) have been identified in glycosomes: ABC transporters
[93] and MCF proteins belonging to the Mitochondrial Carrier
Family [58]. Three so-called half-ABC transporters (called
GAT1-3, for Glycosomal ABC Transporter) have been
identified in the glycosomal membrane of T. brucei, and
homologues were found in the genomes of T. cruzi and L.
major. T. brucei GAT2 has been shown to complement, to a
limited extent, growth on oleate by Saccharomyces cerevisiae
cells deficient in both peroxisomal fatty-acid transporters PAT1
and PAT2 [94]. GAT1 and GAT3 were not able to complement
the deficiency; no information is as yet available about their
substrate specificity. GAT1 is predominantly expressed in
procyclic T. brucei. Interestingly, GAT2 is only expressed in
bloodstream-form T. brucei in which glycolysis is by large the
major function of glycosomes. However, for theoretical
reasons, we consider it doubtful that ABC transporters are
involved in the transport of glycolytic intermediates. The ATP
yield of glycolysis is low: maximally two molecules of ATP
per molecule of glucose consumed. Any further decrease of
this yield by spending ATP for transmembrane transport
processes within the glycolytic process itself would be highly
disadvantageous for the cell, and thus unlikely. We therefore
consider it more likely that other kinds of transport processes
are responsible for the glycolytic intermediates. A first option
is the involvement of MCF transporters. Indeed, trypanosomatid genomes contain several MCF homologues, most of
them probably present in the inner mitochondrial membrane
[87]. But the recent proteomic analysis detected three of them
in glycosome preparations of procyclic T. brucei [58]. However, none has been found so far in glycosome preparations
from bloodstream-form cells. The three MCF molecules found
in procyclic glycosomes present the highest percentage of
identity/similarity to phosphate carriers, dicarboxylate carriers
and ATP/ADP exchangers. However their functional identity
remains to be determined and their glycosomal localization
confirmed.
Several other possibilities for solute translocation through the
glycosomal membrane may be considered. (1) Entry into or exit
from glycosomes by diffusion along the (electro)chemical
gradient via specific transporters or pores not belonging to a
known class of proteins and that remain to be identified (2)
Exchange or antiport processes not involving MCF proteins, in
which the import of one metabolite is obligatory coupled to the
export of another one. Also in this case, the energy would be
provided by the transmembrane gradients of the compounds.
Kinetic and stoichiometric analyses suggest that such exchange
is very likely to happen for the transport of Gly3P and DHAP
[95,96]. Antiport systems for other metabolites could be
imagined as well. (3) A more speculative possibility is an
association of substrate-specific soluble enzymes (in the
glycosomal matrix or cytosol) with non-specific carriers or
pores in the glycosomal membrane, thus creating a kind of
specific ‘group-translocation process’ by which the vectorial
process is driven by its coupling to the enzymatic conversion.
This might, for example, be the case for glucose uptake and its
subsequent phosphorylation by HXK, resembling the PEPdependent phosphotransferase system responsible for glucose
transport in many bacteria [97]. However the glycosomal system
would be fundamentally different from the bacterial one,
because in trypanosomes glucose phosphorylation is performed
by an authentic ATP-dependent HXK. Interestingly, trypanosomatid HXK seems to have affinity for membranes. Furthermore,
it could be imagined that also other enzymes may associate with
such a non-specific pore or transporter, either other sugar-phosphorylating enzymes or enzymes catalyzing entirely different
reactions at the beginning or end of a glycosomal pathway. The
former option may be the case in glycosomes of the fish parasite
Trypanoplasma borreli, where the full set of glycolytic enzymes
could be found except HXK [98]. The fact that the metabolic
schemes presented above invoke many solute translocation steps
through the glycosomal membrane, together with the observed
low protein content of the membranes and the difficulty in
identifying such candidate transporters by classical biochemical
means and modern genomic and proteomic analyses seem to
make it worth to experimentally pursue the latter option.
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7. Role of glycosomes in metabolic programming during
differentiation of trypanosomatids
As described above, the life-cycle of trypanosomatids is
highly complex; the parasites are at every step morphologically
and metabolically well adapted to a distinct compartment of
their specific hosts. As the successive differentiation stages may
encounter highly different environments, the parasites have to
rapidly change their metabolism to cope with the altered
conditions. The compartmentation of environment-sensitive
parts of metabolism within glycosomes could be a way by
which the trypanosomes achieve such flexibility. As explained
in previous sections, the enzymatic content of the organelles
may vary considerably from one adaptive form to the other;
therefore, it could be imagined that a rapid way to adapt the
cell's metabolism is changing its glycosome population by
specifically destroying the old, not anymore adapted organelles
and inducing the synthesis of new ones, similar as has been
reported for yeast peroxisomes when growth conditions change
(Fig. 4).
The peroxisome renewal process has been studied in detail in
methylotrophic yeasts. Glucose-grown Hansenula polymorpha
contains only one or two small peroxisomes. When these cells
are transferred to methanol-containing medium, proteins of
methanol assimilation are imported so the organelles grow and
new organelles are formed by fission from the mature ones
[99,100]. After fission the mature organelles lose the capacity to
incorporate new protein; protein import is confined to the
smaller organelles that have budded off. The other part of the
process is the removal of the redundant organelles. The
degradation of peroxisomes occurs by a process called
11
pexophagy, a specific form of autophagy, as described in detail
elsewhere in this issue (reviewed in [101,102]). Autophagy and
pexophagy share several proteins with the cytoplasm-tovacuole targeting (Cvt) pathway by which some newly
synthesized vacuolar proteins of yeasts are routed to their
organelles. This specific degradation of peroxisomes can occur
by two different ways. The first one, known as micropexophagy, involves the complete engulfment of the organelles by
extended vacuole arms. The second one, called macropexophagy is even more selective: the organelles to be degraded are
surrounded by membranous layers to form an autophagosome,
whose membrane will fuse with that of the vacuole. The
induction of either micro- or macropexophagy depends on the
nature of a nutrient signal and differs between species. By the
combination of selective and induced degradation and synthesis
of peroxisomes, yeasts can create a population of organelles
with a different set of enzymes, and thus adapt efficiently and
rapidly to new environmental conditions.
The situation might be similar for trypanosomatids: the
glycosomes may be heterogeneous; a subset of proliferationcompetent organelles may give rise to a population with
enzymes that allows differentiating parasites to deal with the
new conditions encountered by the new life-cycle stage,
whereas the old organelles could be degraded specifically.
Indeed, recent evidence suggests the presence of autophagy processes in these organisms, particularly during the
differentiation from one life-cycle stage into the next one. First,
the occurrence of autophagy has been reported previously for L.
major [103,104], and our recent database searches using the
three trypanosomatid genomes against 40 autophagy-related
genes (ATG) coding for proteins involved in these processes
Fig. 4. Model of the turnover of glycosomes during different steps of the trypanosome's life cycle. For details, see text. Modified from [108].
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showed that the autophagy machinery is at least partially present
in these organisms [105]. In eukaryotic cells, the Cvt and
autophagy pathways can be divided in different steps including
the induction and signalling, the cargo selection and packaging,
the vesicle nucleation, the retrieval, the docking and fusion, and
finally, the vesicle breakdown [106]. Many of the proteins
involved in these steps are implicated in different cellular
processes, others are specific for one or more forms of
autophagy. For each of the different steps, orthologues were
either confidently identified in at least one trypanosomatid
database or were found to be likely present. Only the Cvt
pathway seemed absent. However, for each step of autophagy
only half of the proteins were identified, suggesting that the
autophagy process in trypanosomatids is much simpler than that
of yeast, but still viable from a genomic point of view since no
key compounds were absent. Recently, autophagy was found to
play a role in the differentiation and acquisition of the virulence
of L. major [107]. An ATG4 null mutant was unable to
differentiate into the infective form. Using a fluorescent ATG8
fusion protein as an autophagosome marker, an increase of
autophagy was monitored during differentiation. These results
confirmed the presence of these two proteins, which were both
classified as ‘likely to be present’ in the genomic survey, as well
as the presence of a specific autophagy structure, the
autophagosomes, in trypanosomatids.
Furthermore, our genomic analysis seemed to indicate that
the parasites possess the molecular machinery to degrade
selectively their glycosomes by a process analogous to either
macro- or micropexophagy. This analysis needs now to be
confirmed experimentally. Our (MH and PM) unpublished
results with T. brucei have revealed that, during the differentiation of this parasite from the bloodstream to the procyclic
form, glycosomes co-localize with enlarged lysosomes and
glycosomal enzymes specific for the bloodstream form
disappear rapidly after differentiation, concomitant with the
synthesis of specific procyclic ones.
Together, the available data support the hypothesis that
glycosomes are rapidly replaced during the transition between
different life-cycle stages of the parasites. This process is
possibly similar to pexophagy in yeasts; it may be simplified
and involve different or additional proteins than the yeast ATG
orthologues found in the genomic search. The compartmentation of the enzymes in glycosomes and the rapid
replacement of these organelles by others with a different
metabolic capacity may therefore augment the adaptability of
the trypanosomes to different environmental conditions. Since
glycosomes may provide this increased metabolic adaptability
to all trypanosomatids and at the various steps when they
transform from one life-cycle stage to the next one, it may be
considered as a highly important function of these organelles.
8. Conclusions
Glycosomes of Kinetoplastida share some metabolic functions with peroxisomes of other organisms, such as defense
against reactive oxygen species and an involvement in etherlipid biosynthesis and β-oxidation of fatty acids, but they are
unique in sequestering the glycolytic pathway, as well as some
other parts of the network for carbohydrate metabolism. This
subcellular organization of metabolism appears to have some
important consequences for these organisms. Proper biogenesis
of glycosomes and correct glycosome-targeting of glycolytic
enzymes is essential for trypanosomes when grown on glucose,
probably because incorrect or incomplete compartmentation
affects the regulation of the glycolysis. Moreover, the
compartmentation enables these organisms to overcome short
periods of anaerobiosis, albeit without sustaining growth.
Interestingly, the enzymatic content of glycosomes may vary
importantly between different life-cycle stages of parasitic
trypanosomatids. Possibly, the sequestering of their pathways
for carbohydrate metabolism within glycosomes enables the
parasites to adapt rapidly to new nutritional conditions during
the differentiation of one life-cycle stage to the next by the ‘en
bloc’ degradation of one population of organelles through
autophagy, concomitant with the synthesis of a new population
of glycosomes with a different enzymatic content. This
glycosome-dependent capacity for efficient and rapid metabolic
adaptation may have played an important role in the successful
radiation of kinetoplastids as parasites with multiple, highly
different niches in one or several host species.
Acknowledgements
The research by PM and VH was supported through grants
from the ‘Fonds de la Recherche Scientifique Médicale’ (FRSM)
of the ‘Communauté française de Belgique’ and the Interuniversity Attraction Poles-Belgian Federal Office for Scientific,
Technical and Cultural Affairs. The research by FB received
support from the CNRS, the Conseil Régional d'Aquitaine and
the Ministère de l'Education Nationale de la Recherche et de la
Technologie. MH acknowledges a PhD scholarship from the
‘Fonds pour la formation à la Recherche dans l'Industrie et dans
l'Agriculture’ (FRIA) and the Université catholique de Louvain.
We are grateful to Dr. Fred Opperdoes for helpful discussions
and critical reading of the manuscript.
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