Eur. J. Biochem. 236, 228-233 (1996)
0 FEBS 1996
Glucose uptake occurs by facilitated diffusion
in procyclic forms of Trypanosoma brucei
Ulrike WILLE', Andreas SEYFANG' and Michael DUSZENKO'
'
Physiologisch-chemisches Institut, Universitat Tubingen, Germany
Molecular Microbiology & Immunology, Oregon Health Science University, USA
(Received 30 October 1995)
-
EJB 95 1775/6
The glucose transporter of Trypanosoma hrucei procyclic forms was characterized and compared with
its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a
saturable uptake process of relatively high affinity (K," = 80 pM, V,,, = 4 nmol min-' lo-' cells), which
showed substrate inhibition at glucose concentrations above 1.5 mM (Kl = 21 mM). Control experiments
measuring deoxy-~-['H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of
Qlo= 2.33 and an activation energy of E, = 64 kJ mol-'. As shown by trans-stimulation experiments,
glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor
studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone(5 pM), the
H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 pM), the ionophor monensin (1 pM), or the
Na'/K'-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally
reconstituted into proteoliposomes. Using Na'-free conditions in the absence of a proton gradient, the
specific activity of D-['4C]glUCoSe transport was determined as 2.9 nmol min-' (mg protein)-' at 0.2 mM
glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of
the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.
Keywords: facilitated diffusion; functional reconstitution ; glucose uptake ; hexose transporter; Trypanosoma brucei.
African trypanosomes cause severe sleeping sickness in human and nagana in livestock throughout the equatorial part of
the continent, threatening millions of people and vastly affecting
meat and diary production. This protozoan parasite undergoes a
complex lifecycle, including distinct stages in both the mammalian host and the insect vector. Vector and bloodstream forms
show marked differences, especially in energy metabolism,
which is restricted to glycolysis in the latter forms, whereas insect forms possess a well developed mitochondrion and express
a functional citric acid cycle as well as the respiratory chain.
Hence, glucose uptake is pivotal to bloodstream-form metabolism but seems only marginally important for procyclic vector
forms. Nevertheless, procyclic-form parasites metabolize glucose from the medium and we thus questioned whether the respective glucose transporter is different from that expressed in
bloodstream forms, i.e. if expression of the glucose carrier is
differentially regulated during the lifecycle. This question
seemed intriguing, since conflicting results have been published
previously. Whereas earlier data suggested that glucose uptake
is protonmotive-force driven [l],indirect results obtained in a
Correspondence to M. Dusrenko, Physiologisch-chemisches Institut,
Universitlt Tubingen, Happe-Seyler-Str. 4, D-72076 Tubingen, Germany
Fax: +49 7071 296390.
Abbreviations. E,, activation energy; Ql0, temperature quotient;
THT, trypanosome hexose transporter.
Enzymes. Hexokinase (EC 2.7.1.I); glucose-6-phosphate dehydrogenase (EC 1.1.1.49).
chemostat culture suggested a facilitated diffusion mechanism
[2]. Additionally, genetic experiments proposed a similarity of
about 80% for both the procyclic-form and the bloodstreamform glucose carriers [3]; the latter, however, was clearly shown
to work by facilitated diffusion [4, 51.
Procyclic vector forms reside mainly in the midgut of the
tsetse fly, which feeds exclusively on the blood of higher vzrtebrates. Glucose may thus be abundantly available immediately
after a bloodmeal, but will be limited in between feeding intervals. Proline is extensively used as an energy supply by all
species of the genus Glossina, where it is a major constituent of
the haemolymph (up to 2%) [6]. Procyclic trypanosomes also
depend on proline as an energy supply and express a high activity of proline oxidase [7], but only one of two possible trypanosome hexose transporter (THT) genes, THT-2. In contrast. the
bloodstream form with its exceptionally high need for glycolyzable substrates expresses both genes (THT-I and THT-2) in a
ratio of 40:1, as judged by the corresponding mRNA levels [ 3 ] .
In this study, we characterize a high-affinity, facilitated diffusion glucose transporter of the procyclic insect stage and compare it with a transporter isoform of bloodstream forms. A preliminary account of this work was presented earlier [8].
MATERIALS AND METHODS
Trypanosomes. Procyclic forms of Trypanosoma hriicei,
derived from bloodstream forms of MITat 1.4 were cultured at
Wille et al. (Eur: J. Biochem. 236)
27°C in modified minimum essential medium 171, containing
fetal calf serum (lo%), proline (5.2 mM), pyruvate (2 mM), glutamine (2 mM) and hemin ( 3 1.5 pM). The incubation medium
used for uptake studies lacked these supplements but included
various D-glucose concentrations according to assay conditions.
Preparation of cells for uptake studies. Trypanosomes
were taken from logarithmically growing cultures and centrifuged for 10 min (1500Xg; 4°C). The cell pellet was washed
twice in incubation medium and finally resuspended in the same
medium to a cell density of 5X108 trypanosomes ml-'. Cells
were transferred to a shaking water bath (27°C) and uptake
studies were performed after a 5-min incubation period.
Glucose and protein determination. Glucose concentration
was determined enzymatically, using hexokinase and glucose-6phosphate dehydrogenase, as described earlier [4] ; all measurements were performed in triplicate. Total cell protein was determined using a photometric assay (Bio-Rad protein assay, BioRad Laboratories) with bovine serum albumine as standard
protein.
Glucose uptake assay. Cells were incubated in the presence
of different inhibitors or ionophores at a glucose concentration
of 5 mM. An incubation was performed for 5 min at 27°C with
or without inhibitors, and the actual glucose concentration at
time-point zero was determined in each case. During the
following 1-h incubation, 200 pl aliquots were withdrawn at
10 min intervals and centrifuged for 30 s each in a Beckman
microfuge (13000Xg; 20°C). 150 p1 of the supernatant was
thoroughly mixed with 7.5 pl ice-cold perchloric acid (70%, by
vol.) and stored for up to one week at 4°C until glucose was
determined. Inhibitors or ionophores were dissolved in either
ethanol [phloretin, phlorizin, monensin, carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone, and nigericin], 1: 1 ethanol/
Me,SO (cytochalasin B), or water (ouabain and N,N'-dicyclohexylcarbodiimide). Mock treatment of trypanosomes with these
solvents alone served as controls. To incubate trypanosomes in
the absence of Na+, cells were washed twice and finally resuspended in sodium-free buffer. In order to restore the osmolarity
in this medium, all Na' salts were replaced by their K+-salt
analogues.
2-Deoxy-glucose uptake assay. Trypanosomes from logphase culture were washed twice in 120 mM NaCI, 10 mM sodium phosphate, pH 7.4 (NaCIP,) and resuspended in prolinefree MEM at a concentration of 1X 1Ox cells ml I . After incubation for 5 min at 25"C, 180 pl cell suspension was added to
20 p1 deoxy-~-[~H]Glc
(0.5 pCi, Amersham) at various concentrations in a range 0.01-10 mM. After 10 s, the uptake was
stopped by centrifugation, spinning the cells through an oil cushion of 100 p1 3 -bromododecane (d = 1.038 g/ml, Sigma) in Eppendorf tubes. Tubes were snap-frozen in a methanol/dry ice
bath immediately after centrifugation, before the cell-pellet-containing tip of the vial was clipped off and mixed with 200 pl
1% SDS. Incorporated radioactivity was determined by liquid
scintillation counting.
Trans-stimulation of glucose transport. The stereospecificity of glucose transport was determined by truns-stimulation experiments. For this purpose, 90 p1 cell suspension
(5X1OX trypanosomes ml-l) were loaded for 8 min with 10 pl
deoxy-~-[~H]Glc
(17 Ci mmol-'; Amersham), before 10 p1 different non-labelled sugars (D-glucose, L-glucose, D-fructose ; final concentration, 20 mM each) were added. Cells were incubated for various times and de~xy-D-[~H]Glc
efflux was stopped
by collecting the cells onto nitrocellulose filters (0.45 pm) during filtration of the suspension. The filters were washed three
times with 3 ml ice-cold NaCl/P,, placed in 5 ml scintillant ULTIMA GOLDTM(Packard) and analyzed by liquid scintillation
counting.
229
Functional reconstitution of the procyclic glucose transporter. The solubilized and concentrated membrane fraction was
reconstituted in sonicated liposomes by the freezekhaw-sonication method previously described 151. Briefly, trypanosomal
ghosts from 10'" procyclic cells (equivalent to 8 mg protein)
were treated with EDTA at pH 12 to remove cytoskelctal and
membrane-associated proteins. The resulting 230 pg EDTNalkali treated vesicles were incubated with the non-ionic detergent
octylthioglucoside which solubilized about 50 % of the membrane proteins. Subsequent removal of the detergent by dialysis
and concentration by ultrafiltration yielded about 94 pg plasmamembrane proteins, i.e. a similar yield and enrichment as obtained for bloodstream forms [ 5 ] .Reconstituted proteoliposomes
were resuspended in 30 mM Tris/HCI, pH 7.4, containing 2 mM
MgCI, at a concentration of 7-10 pg protein and 330 pg phospholipids/lOO 1-11 transport assay. Transport was measured at
25 "C and initiated by the addition of 10 p1 radiolabelled glucose
(0.4 pCi ~-[l-'"C]glucoseor ~ - [ l - ~ ~ C ] - g l u c o52
s e ,Ci mol-' or
57 Ci mol-' respectively, purchased from Amersham) at 0.2 mM
final concentration to 90 p1 reconstituted proteoliposomes. Uptake of L-glucose served as control for non-specific adsorption
or membrane permeability. Transport was stopped after 15-60 s
by addition of 3 ml ice-cold stopping buffer (30 mM Tris/HCI,
2 mM MgCl,, 200 pM phloretin, pH 7.4) and immediately
filtrated through a nitrocellulose membrane filter (0.2 pm ; Sartorius). Following two additional washes of the filter with the
same buffer, glucose uptake into proteoliposomes was monitored
by liquid scintillation counting.
RESULTS
Kinetics and temperature dependence of D-glucose uptake.
Glucose uptake by procyclic forms of 7: brucei was measured at
27°C with 0.15-9 mM D-glucose during the linear uptake period (up to l h). Glucose uptake showed a saturable kinetic typical for a carrier-mediated process (Fig. 1). A channel-mediated
uptake was excluded due to the truns-stimulation properties of
transport. Using regression analysis (Lineweaver-Burk), we determined an apparent K,,, of 80.01 ? 15.32 pM and a V,,, of
4.29 -f 0.51 nmol min- I 10 -'cells, equivalent to 0.7 mg total
protein (Fig. 1A). Additional experiments measuring deoxy-D['HIGlc uptake for 10 s under zero-trans conditions were used to
determine the uptake kinetics for the concentration range 0.01 1 mM, which was inaccessible for enzymatic measurements.
These experiments revealed an apparent K,,, of 75 pM (Fig. 1B),
which is in good agreement with the value obtained for D-ghcose. In contrast to other well-characterized glucose transporter
systems, including the bloodstream-form carrier, however, sugar
concentrations above 1.5 mM led to a substrate inhibition of this
transporter. The inhibitor constant was determined as K, =
21.32 ? 4.22 mM, which indicates that this inhibition may be
irrelevant at physiological conditions. Interestingly, deoxy-D['HIGlc uptake showed no substrate inhibition within the concentration range tested (0.01 -10 mM; data show the range
0.01 - 1 mM, Fig. 1B) and revealed a K,,, similar to the value
obtained for D-ghCOSe (see above). Since 2-deoxy-D-glucose is
phosphorylated by hexokinase but not further metabolized, we
conclude that the observed substrate inhibition in procyclics occurred at the level of glycolysis after the hexokinase reaction
and, hence, affected glucose transport only indirectly. The glucose transporter was further characterized by temperature dependent uptake studies at 20-33°C (Fig. 2). From these data, the
temperature quotient [Q," = (R2/R,)""'1 ',. , T2 > TI] was calculated; Ql0 = 2.33 ? 0.02. The activation energy was determined
from the negative slope of Fig. 3 as E, = 64.01 L 2.25 kJ mol~( r = -0.9917).
230
Wille et al. (Eur J. Biochem. 236)
4
e
0.40
70
0.35
4
x
0.30
4
0
fi 0 . 2 5
a
v
0.20
0
Y
4
$
0.15
a,
0.10
0
9
0.05
0.00
0
20
10
30
50
40
60
time (min)
Fig. 2. Temperature-dependent glucose uptake. Cells were incuhted
for 1 h at 20, 23, 27, 30 and 33°C in a modified minimum essential
medium containing 5 mM D-gluCose. Assays were performed as described in Fig. 1 (triplicate measurements of three different experiments).
A temperature quotient of Q,,, = 2.33 5 0.02 was calculated from the
different glucose uptake rates.
1
0.0
0.0
B
'
I
I
0.5
1.0
[Z-deoxy-D-glucose]
1
I
(mM)
Fig. 1. Kinetics of D-glucose and 2-deoxy-~-glucoseuptake. Procyclic
culture-form trypanosomes were washed twice in a modified minimum
essential medium and resuspended to a density of 5X10xcellsml-'. Prior
to the uptake assay, cells were incubated for 5 min under experimental
conditions. (A) D-glUcose uptake under equilibrium-exchange conditions
[20].200 p1 aliqots were taken in 10-min intervals during the linear uptake period (up to 60 min). Cells were immediately pelleted by centrifugation, using the supernatant to enzymatically determine the remaining
glucose concentration (triplicate measurements of three independent experiments). (B) Deoxy-~-['H]Glc uptake under zero-trans conditions
[20].The uptake assay was initiated by adding radiolabelled 2-deoxy-Dglucose to the cells and terminated after 10 s by spinning cells through
an oil cushion. lncorporated radioactivity was determined within the cell
pellet by liquid scintillation counting (two independent experiments).
Michaelis-Menten analysis: K,, and V were calculated by:
x mi
2.0
s
h
7
1.6
0
4
1.6
4
I
;.
1.4
1.2
I
0
-g
*d
1.0
0.8
.4
0.6
I
3 .90
3.95
I
I
4.00
1/RT (lO-'x
I
I
4.05
I
I
4.10
,
4.15
mol x J-')
Fig.3. Arrhenius plot of glucose uptake rates at 20-33°C. From
Fig. 2 the In u of glucose uptake was calculated for different incubation
temperatures and plotted against R-' T ' _ The negative slope of the
graph represents the activation energy (E., = 64.01 ? 2.25 kJ mol-' ; 11 =
31.
Glucose uptake was not significantly affected by any of the
used inhibitors (Table 1). Neither sodium-free incubation nor
the Na+/K+-ATPase inhibitor ouabain (1 mM) or the ionophore
monensin (1 pM) reduced the uptake rate profoundly. Additionally, inhibition of sugar transport by the H'/ATPase-inhibitor
Inhibitor studies of glucose uptake. The mechanism of D- 20 pM N,N'-dicyclohexylcarbodiimide, the ionophore carhonglucose uptake was determined by incubating trypanosomes in ylcyanide-4-(trifluoromethoxy)phenylhydrazone (1 pM m d
the presence of different inhibitors, which are known to interfere 5 pM) or nigericin (3 pM) was insignificant. From these data.
with distinct classes of hexose transporters such as facilitated we conclude an ion-independent D-glUCoSe transport in procyclic
diffusion (cytochalasin B, phloretin and partly phlorizin), Na+/ forms of 7: brucei. In contrast to the carrier in bloodstream
glucose symport (monensin, ouabain, Na+-free conditions, and forms [4] and human erythrocytes [9, lo], the glucose transporphlorizin) and Hi /glucose symport [N,N'-dicyclohexylcarbo- ter of the procyclic form was also insensitive to cytochalasin B
diimide, carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone and phloretin. Even high inhibitor concentrations (up to 300 pM)
and nigericin; see [4] for details]. Since some of the inhibitors showed only insignificant effects on glucose uptake. Finally,
are insoluble in water, mock experiments with the respective phlorizin, a specific inhibitor of intestinal and renal sodium-desolvent (0.55% Me,SO, 0.5 % ethanol) in mediator-free media pendent glucose uptake in mammals [ll, 121 and partly of the
were performed but had only negligible effects on the parasites facilitated diffusion pathway I l l ] reduced glucose uptake o n l y
by 2%, i.e. within the range of standard deviation.
(data not shown).
23 1
Wille et al. ( E m J. Biochem. 236)
Table 1. Comparison of the glucose transporters from Z brzicei bloodstream and procyclic forms. Data were determined at 37°C for bloodstream forms and at 27°C for the procyclic culture forms. Data for bloodstream forms were from [4]. Potential inhibitors or ionophores of the three
different eukaryotic glucose-transport systems including facilitated diffusion (l),Na'lglucose symport (2) and H+/glucose symport (3) were used.
K,
Form
V
Qio
Glucose uptake
E.4
cyt. B (1)
300 pM
' mg-'
PM
nmol min
Bloodstream
Procyclic
490 i 140
80.0 -+ 15.3
252
5 43
6.13 i 0.73
Form
ouabain (2)
inonensin (2)
1 mM
1 PM
2.0 i 0.2
2.3 i- 0.02
Na+-free (2)
kJ mol- '
%
52.1 t 1.0
64.0 5 2.25
23 i 6
98 i 9
phloretin (1)
100 pM
phlorizin (1,2)
500 FM
40 i 4
92 5 6
86 i 4
98 i- 7
N,N'-dicyclohexylcarbodiimide (3)
20 pM
carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (3)
nigericin (3)
1 PM
5 PM
3 PM
95 i 2
97 i- 6
95 i- 3
96 i 3
-
-
81 i 1
87 i- 4
%
Bloodstream
Procyclic
100 -+ 5
98 i 4
92 i 2
89 i 5
101 5 5
100 i 8
2000
1500
1000
500
li
0
2
4
6
8
10
12
14
16
time (min)
Fig. 4. Trans-stimulation of deoxy-~-[~H]Glc
uptake by D-glucose, Lglucose and D-fructose. After a 5-min incubation at 2 7 T , 5X10xcells
ml ' were loaded for 8 rnin with deoxy-~-[~H]Glc.
Appropriate sugars
(o-glucose, L-glucose, D-fructose; 20 mM each) were added and trypanosomes were further incubated for different times. Deoxy-~-[~H]Glc
efflux was terminated by filtering the cell suspension through a nitrocellulose membrane (0.45 pM). Cells were washed three times with 3 ml
ice-cold NaCI/P, and radioactivity was determined using a liquid scintillation counter. Values are the means of three independent experiments.
Stereospecificity of glucose transport. The stereospecificity of
glucose uptake in the procyclic form of 7: hrucei was measured
by trans-stimulation experiments, using deoxy-~-['HH]Glc,a glucose analogue which is taken up and phosphorylated by hexokinase, but cannot be further metabolized. After loading the cells
for 8 rnin with deoxy-~-['HlGlc,unlabeled D-glucose, L-glucose
or D-fructose were added to the incubation medium at a final
concentration of 20mM. Assuming that these sugars use the
same carrier system, an efflux of deoxy-D-('H]Glc is to be expected [13, 13al and intracellular radioactivity should be released. Our data show that L-glucose could not efficiently compete with the glucose transporter, whereas both D-glucose and
D-fructOse led to an increasing deoxy-~-[~H]Glc
efflux (Fig. 4).
These results suggest that D-glucose, D-fructose and 2-deoxy-D-
0
15
30
time
45
60
75
(9)
Fig. 5. Functional reconstitution of procyclic glucose transporter in
liposomes. The glucose transporter-enriched protein fraction (ultrafiltrate of a detergent-solubilized plasma-membrane fraction) was reconstituted into sonicated proteoliposomes by a freezelthaw-sonication method
as described [5]. The transport conditions were sodium-free and contained no proton gradient. Uptake of ~-['~C]gIucose
and ~~-['~C]gIucose
as controls were measured at 25 "C and stopped with ice-cold buffer by
rapid filtration through a membrane filter.
glucose use the same carrier system and that the hexose transporter is stereospecific for the D isomers. Additionally, the
deoxy-~-['H]Glc efflux was higher with D-fructose than with Dglucose, although the transporter seems to have a 20-fold lower
affinity for fructose [1] than for glucose. This discrepancy may
be explained by a higher turnover number for fructose or a faster
phosphorylation of glucose, resulting in different intracellular
concentrations of non-phosphorylated hexoses.
Functional reconstitution of the procyclic D-glUCoSe transporter. A D-glucose-transporter-enriched plasma-membrane
fraction was reconstituted in liposomes and D-glUCOSe-SpeCifiC
transport into the resulting proteoliposomes was determined by
measuring the D-ghCOSe uptake minus the unspecific L-glucose
transport, which was monitored as a negative control (Fig. 5 ) .
232
Wille et al. (Eur: J. Biochem. 236)
The specific activity of D-glucose uptake was 2.9 nmol min-'
(mg protein)-' at 0.2 mM glucose and 25°C. This value was
higher than the D-ghcoSe-Specifk transport of the bloodstream
form under identical reconstitution conditions [1.9 nmol min-'
(mg protein)-'] [5], and is in good agreement with the higher
substrate affinity of the procyclic glucose transporter (Table 1).
Since the reconstitution system was sodium-free and contained
neither a proton gradient nor ATP, these results give additional
support for a facilitated-diffusion glucose transporter in the
plasma membrane of procyclic trypanosomes. Furthermore, the
transport activity of the reconstituted procyclic glucose transporter corresponds well with D-glucose-specific transport activity reported for the erythrocyte glucose transporter after reconstitution of Triton-solubilized membrane fractions [14, 151.
DISCUSSION
Unless a specific transfection pathway exists, e.g. during
sexual intercourse or by crossing the placental barrier, transmission of a protozoan parasite from one host to another depends on
a vector for transportation. Most often, these vectors are bloodsucking arthropods, allowing the parasite to survive for prolonged periods of time. Parasites, such as African trypanosomes,
live within at least two completely different but equally hostile
environments, such as the mammalian blood with all its immunological phenomena, and the midgut of a tsetse fly with all
its digestive capacities. The ultimate interface between host and
parasite is the cell membrane of the latter, which must be rigidly
organized to withstand all lytical attacks of the different hosts,
but permeable enough to allow intracellular homeostasis of metabolites and ions. Bloodstream forms of African trypanosomes
therefore contain a densely packed surface coat, consisting of
about 10' virtually identical molecules of a variant surface glycoprotein, which protects the cell against the immune defense,
except specific antibodies formed during the infection. Variant
surface glycoprotein, however, is subject to antigenic variation,
thus ensuring a persisting parasitemia [16]. Upon ingestion by a
tsetse fly, the parasite transforms to the procyclic vector form,
thereby shedding variant surface glycoprotein molecules and replacing them with procyclic acidic repetitive protein, to be able
to survive within the midgut of the insect. This process is paralleled by the formation of a functionally active mitochondrion
[ 17, 181, the switching from substrate-level phosphorylation during glycolysis to oxidative phosphorylation during the electrontransport chain, and, obviously, by the differential expression of
carriers and maybe ion channels. Since so far very little information is available about different substrate transporters expressed
in either form, we were prompted to functionally characterize
the glucose transporter of procyclic forms and to compare it with
its bloodstream form analogue.
Our results show marked differences between the hexose
transporters expressed in procyclic and in bloodstream forms.
Most surprisingly, whereas the bloodstream-form transporter
was significantly inhibited by both cytochalasin B and phloretin
(indicative of facilitated diffusion) (4, 9, 101 glucose uptake in
procyclic trypanosomes has not been inhibited by any compound
used so far. Nevertheless, the kinetic data clearly show that glucose uptake in the latter form is also carrier mediated. Whereas
Na+/glucose or H+/glucose symport is specifically inhibited by
ouabain, monensin, phlorizin and Na' -free media or by N,N'-dicyclohexylcarbodiimide, carbonyl-4-(trifluoromethoxy)phenylhydrazone and nigericin respectively, inhibition of facilitated
diffusion by phloretin is rather unspecific, probably depending
on the transporter geometry and its accessibility on the membrane. Functional reconstitution of the procyclic glucose trans-
porter in proteoliposomes was Na' independent and did not require a proton gradient. Based on our results, we thus favour a
facilitated-diffusion uptake mechanism for hexoses in procyclic
forms, similar but not identical to that for bloodstream forms.
Both forms of the transporter are stereospecific and also accept
fructose as a substrate. Interestingly, however, the primdry
bloodstream form transporter (THT-1) showed a sixfold lower
substrate affinity as the procyclic transporter (Table 1). It should
be noted, however, that the glucose concentration is invariably
high in blood and thus a high affinity is not a requisite for a
sufficient substrate supply, as it might be in the lumen of the
insect midgut. Together, expression of a more effective transport
system i n the insect form seems to correlate with the environmental conditions. The observed substrate inhibition of the glucose transporter in procyclics, induced by glucose concentrations
above 1.5 mM, appears to be at the metabolic level rather than
at the transporter site, since we used equilibrium-exchange conditions to measure glucose uptake. If 2-deoXy-D-ghcose was
used in 10-s uptake assays (zero-truns conditions), a substrate
inhibition was not observed.
Earlier reports suggested a protonmotive-force-driven glucose uptake in procyclic trypanosomes [I], which was questioned, however, by indirect results using chemostat cultures to
grow procyclic trypanosomes [2]. Further experimental evidencc
against a H+/glucose symport came from a genetic study, showing that bloodstream-form parasites express two hexose transporters (THT-1 and THT-2), from which only one (THT-2) is
expressed in procyclic forms [3]. Since both isoforms posstss
about 80 9
i similarity, the existence of two completely different
transport mechanisms seems rather unlikely. Our results indeed
favour facilitated diffusion, suggesting that both transporters are
distinct in terms of kinetic parameters, but similar i n terms of
the uptake mechanism. However, the experimental conditions
were strikingly different in both studies; whereas Parsons ;tnd
Nielsen [ 11 used radiolabelled 2-deoxy-~-glucoseat low conczntration (30 pM) and terminated the assay after 30 s, we primarily
usGd D-ghCOSe at high concentrations (5 mM) and monitored
glucose transport during the linear uptake period (up to 60 min).
We, thus, cannot rule out the possibility that the procyclic hzxose transporter may work as an active ion-dependent carrier at
low substrate concentration but uses a facilitated diffusion process if glucose is readily available. The phenomenon of substrate
transport by ion symporters without the counterion has been described for some i o n h g a r transporters and was termed 'slippage' [ 131. Alternatively, procyclic trypanosomes may possess a
facilitated-diffusion glucose transporter in the plasma msmbrane, but an active H '/glucose symporter in the glycosonies
1191.
We thank Dieter Mecke and Egbert Geyer (University of Marburg.
Germany) for helpful discussions. This work was supported by a research grant from the Deutsche For.~chungsgemein.scha~c
(DFG).
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