Characterization of glycerol uptake in bloodstream and procyclic forms of <i>Trypanosoma brucei</i>

Wille, Ulrike; Schade, Babette; Duszenko, Michael

doi:10.1046/j.1432-1327.1998.2560245.x

Cited by 14 publications

(12 citation statements)

References 29 publications

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“…We have shown previously that glycerol uptake is consistent with facilitated diffusion through a transporter protein (10). This investigation gave clear evidence for an additional nonsaturable channel, which remained active after inhibition of the transporter.…”

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confidence: 69%

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Uzcátegui

Szallies

Pavlovic-Djuranovic³

et al. 2004

Journal of Biological Chemistry

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Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production. Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite. As we have shown previously, glycerol transport in T. brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M. (1998) Eur. J. Biochem. 256, 245-250). Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1-3), which show a 40 -45% identity to mammalian AQP3 and -9. AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules. Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs. For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes. In the yeast fps1⌬ mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels. Under iso-and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins. TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone. Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol. Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T. brucei. This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.

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confidence: 69%

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Uzcátegui

Szallies

Pavlovic-Djuranovic³

et al. 2004

Journal of Biological Chemistry

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“…Although glycerol can diffuse across membranes, there are some indications that a plasma membrane carrier can also play a role in its transport in trypanosomes (32). If glycerol could not exit the glycosome sufficiently fast, it might exert a mass-action effect, driving glucose metabolism almost completely toward pyruvate production, as was observed for our GIM5A/B-depleted cells (Table II).…”

Section: Figmentioning

confidence: 69%

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Voncken¹,

Hellemond

Pfisterer³

et al. 2003

Journal of Biological Chemistry

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Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.Microbodies are single-membrane-bound organelles found in very diverse eukaryotes; examples include the peroxisomes of mammals and yeast, the glyoxysomes and peroxisomes of plants, and the glycosomes of kinetoplastid protists. Microbodies manifest considerable diversity with regard to the enzymes and metabolic pathways they contain: examples include catalase, and enzymes of fatty acid beta-oxidation, alcohol oxidation, ether-lipid biosynthesis, and, in the kinetoplastids, glycolysis and glycerol metabolism.

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“…S1) and different kinetic parameters for glycerol uptake. Incorporating the glycerol uptake rate measured in procyclic forms (27) would considerably diminish the predicted Gly3P accumulation (data not shown).…”

Section: Discussionmentioning

confidence: 97%

Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes

Haanstra

Tuijl

Kessler

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

121

146

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ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation. Fig. S1]. In glycolysis, ATP is first invested in 2 phosphorylation reactions, catalyzed by hexokinase (HXK) and phosphofructokinase (PFK). Only further downstream in the pathway the ATP is regained and then a surplus of ATP is generated. In glycerol metabolism, ATP is invested in the reaction catalized by glycerol kinase (GK) (Fig. 1).In the absence of specific regulation of HXK and PFK, the ATP produced by glycolysis could boost the flux through these enzymes above the capacity of the enzymes downstream, and hexose phosphates [glucose 6-phosphate (Glc6P), fructose 6-phosphate (Fru6P) and fructose 1,6-phosphate (Fru1,6BP)] would accumulate to extreme levels. By analogy to the turbo engine (which uses engine exhaust to boost performance), this property was called the ''turbo-design''of glycolysis (2). Many organisms avoid the negative side effects of the autocatalytic design of glycolysis by a tight feedback regulation of HXK and PFK, e.g., inhibition of HXK by Glc6P (3) or trehalose 6-phosphate (5). In yeast, deletion of trehalose-6-phosphate synthase leads to glucose toxicity, accumulation of hexose phosphates and rapid consumption of ATP (4). This ''turbo-explosion'' phenotype is rescued by reducing the expression of HXK (5) or glucose transporters (6).Trypanosoma brucei, the tropical parasite that causes the deadly African sleeping sickness, lacks feedback regulation of HXK and PFK (7-9). The parasite has a complex life cycle that alternates between insect and mammalian hosts; in the latter it lives in the bloodstream, supplied with a ready source of glucose. How then are trypanosomes protected against a possible turboexplosion of glycolysis?A key feature of trypanosome glycolysis is the compartmentation of the first 7 enzymes of glycolysis and 2 involved in glycerol metabolism inside peroxisome-like organelles called glycosomes (10). In the glycosome, ATP and redox levels are ba...

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Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei

Cited by 14 publications

References 29 publications

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes

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