Subcellular Compartmentation of Glycolytic Intermediates in Trypanosoma brucei

Abstract

In cell -fractionation experiments most of the glycolytic enzymes in bloodstream forms of Trypanosomu brucei are recovered in a microbody, called the glycosome [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360 -3641. To see whether this compartmentation of glycolytic enzymes is accompanied by compartmentation of metabolites we have pulse-labelled intact T. brucei with [U-'4C]glucose and followed the incorporation of radioactivity into glycolytic intermediates separated by anion-exchange chromatography. The kinetics of incorporation provide direct evidence for the existence of two pools of glycolytic intermediates. One pool is completely labelled within 15 s and represents 20-30 of total cellular metabolites. Radioactively labelled pyruvate is already produced after I5 s. Since this pool is directly involved in the glycolytic flux, we conclude that it is present in the glycosome. The second pool which represents 70-800/, of the total appears not to be directly involved in glycolysis. Its content equilibrates relatively slowly with the glycosomal pool. It probably represents the cytosol.Incorporation of radioactivity into the total glycerol 3-phosphate pool is more rapid than for the other metabolites studied. This indicates rapid mixing of the glycosomal and cell-sap pools of glycerol 3-phosphate, as required for the extra-glycosomal oxidation of glycerol 3-phosphate by the mitochondria1 oxidase.In the presence of 1 mM salicylhydroxamic acid, which mimics anaerobiosis, the trypanosome produces equimolar amounts of pyruvate and glycerol. Labelling of glycerol and glycerol 3-phosphate proceeds at identical rates but at all times the specific activity of glycerol is less than that of glycerol 3-phosphate. This is compatible with our earlier proposal that glycerol is made from glycerol 3-phosphate by glycerol kinase.We conclude that glycolysis in trypanosomes takes place in the glycosome and that the membrane of this organelle is poorly permeable to most glycolytic intermediates.African trypanosomes, like Trypunosoma brucei, are unicellular flagellate protozoa, that are well adapted to life in the mammalian bloodstream. For their energy needs these organisms rely entirely on glycolysis, which proceeds at an extremely high rate and leads to pyruvate excretion as the only end-product of glycolysis under aerobic conditions [l 1.The NADH formed in glycolysis is reoxidized by means of a mitochondria1 glycerol-3-phosphate oxidase and molecular oxygen. When this oxidase is completely inhibited by 1 mM salicylhydroxamic acid, the trypanosome manages to survive by converting glucose into equimolar amounts of glycerol and pyruvate with concomitant synthesis of ATP.A unique aspect of the glycolytic pathway in T. brucei is that part of it is located inside a microbody-like organelle called glycosome. We have previously shown [2 -51 that nine enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate, are almost exclusively contained in the glycosome. All glycosomal enzymes exhibit high latency in...

Specificity of glucose transport in Trypanosoma brucei

“…Subsequent investigations, in which the production of radiolabeled pyruvate could be detected within only 15 sec indicate labeled metabolites are likely to be problematic in such experiments, perhaps leading to inaccurate quantifications (Visser et al, 1981). This idea is further supported by the higher apparent K,,, obtained from samples taken after 2 min (1.4-1.7 mM) than after 1 min (0.96 mM, Gruenberg et al, 1978).…”

“…This method is not complicated by radiolabeled glycolytic metabolites resulting from the high glycolytic flux in bloodstream forms (Visser et al, 1981). An alternative method, the use of radiolabeled non-metabolizable D-glucose analogues in earlier studies (Gruenberg et al, 1978;Game et al, 1986;Eisenthal et al, 1989), leads to complications by the requirement of adding D-glucose to the incubation medium, since T. brucei bloodstream forms depend exclusively upon glycolysis of exogenous sugar as their sole energy supply.…”

Purification and Characterization of the Native and the Recombinant Leishmania Mexicana Glycosomal Glyceraldehyde‐3‐Phosphate Dehydrogenase

“…However, verification of this by direct measurement of the intra-glycosomal metabolite concentrations is not possible. Trypanosomatids contain 50-250 small glycosomeskell, together making up only 4-5% of the cellular volume, and the coenzymes and metabolites are also present in the much larger cytosolic volume [5]. Therefore, an indirect approach has to be followed, such as studying how a protein with a 'normal', high affinity will function within the glycosome.…”

Glycolysis in Bloodstream Form Trypanosoma brucei Can Be Understood in Terms of the Kinetics of the Glycolytic Enzymes

“…across the plasma membrane and the glycosomal membrane, were lumped into one, because in kinetic experiments no distinction between them has been made so far (23). The glycosomal membrane was taken to be impermeable to metabolites (24), except for those that need to be transported across this membrane (Fig. 1).…”

“…This should also exclude the possibility of a high osmotic pressure in the glycosomes in case of a drop of Gly-3-P and DHAP. Glycosomal concentrations of Glc-6-P (4.4 mM), Fru-6-P (2.4 mM), Fru-1,6-BP (1.9 mM), DHAP (7.1 mM), GA-3-P (0.47 mM), and 1,3-BPGA (0.77 mM) have been estimated (45) on the basis of pulse-chase experiments (24). Except for [Fru-1,6-BP], these concentrations are 1 order of magnitude higher than the concentrations predicted by the model.…”