Abstract
BackgroundGlycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.Methods/Principal FindingsWe hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl−∼0.31), while the other two types of channels are slightly selective for cations (P K+/P Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore.Conclusions/SignificanceThese results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.
Highlights
Trypanosoma brucei is a parasite that belongs to the Trypanosomatidae family of the Kinetoplastea order of protists
In the glycosomal preparations we found only traces of the activities of markers for mitochondria (FADdependent glycerol-3-phosphate dehydrogenase) and flagellar membranes, indicating a low contamination of glycosomes by membrane fragments of these organelles which may be considered as the main potential sources of the contaminating channel-forming activities (Figure 1A, panel c)
As shown by a careful analysis of the images, these structures are not connected to the flagellar membranes, which corroborate the data obtained in the enzyme activity assays performed for the flagellar membrane marker, acid phosphatase
Summary
Trypanosoma brucei is a parasite that belongs to the Trypanosomatidae family of the Kinetoplastea order of protists. As has been shown recently [10], compartmentalization of glycolytic enzymes within the glycosomes of Trypanosomatidae prevents from the detrimental ‘turbo effect’ of an uncontrolled consumption of ATP at the initial steps of glycolysis This is apparently achieved by formation of the two pools of ATP – glycosomal and cytosolic. Considering the evolutionary relationship of peroxisomes and glycosomes one can predict the presence of channel-forming proteins in the glycosomal membrane These channels may allow an easy movement across the membrane of small solutes, including all intermediate metabolites of glycolysis, but prevent transfer of ATP and cofactors and promoting the maintenance of separate pools of these compounds in the cytosol and the glycosomal lumen. Three distinct activities were detected and their electrophysiological characteristics were analyzed using the planar lipid bilayer technique
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