Abstract
In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite’s glycosomes.
Highlights
Whereas in almost all eukaryotic organisms glycolysis is a process that occurs in the cytosol, in Kinetoplastea the major part of the pathway is localized in organelles called glycosomes
The NADH formed in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is re-oxidized inside the organelles by reduction of oxalocetate to malate by a glycosomal malate dehydrogenase (MDH) and in a subsequent reduction of fumarate produced from the malate to succinate by a soluble NADH-dependent fumarate reductase (FRD) [3]
Figure SI (Supplementary Information) shows that most glycosomes were recovered in fractions corresponding to densities of 1.23 to 1.24 g. cm−3 after the isopycnic ultracentrifugation (HK activity was used as glycosomal marker)
Summary
Whereas in almost all eukaryotic organisms glycolysis is a process that occurs in the cytosol, in Kinetoplastea the major part of the pathway is localized in organelles called glycosomes. The NADH formed in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is re-oxidized inside the organelles by reduction of oxalocetate (produced from PEP by PEPCK) to malate by a glycosomal malate dehydrogenase (MDH) and in a subsequent reduction of fumarate produced from the malate to succinate by a soluble NADH-dependent fumarate reductase (FRD) [3]. Another route that may contribute to the regeneration of glycosomal NAD+ involves a NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) [4] that catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (Glyc3P).
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