Environmentally Regulated Glycosome Protein Composition in the African Trypanosome

Abstract

Trypanosomes compartmentalize many metabolic enzymes in glycosomes, peroxisome-related microbodies that are essential to parasite survival. While it is understood that these dynamic organelles undergo profound changes in protein composition throughout life cycle differentiation, the adaptations that occur in response to changes in environmental conditions are less appreciated. We have adopted a fluorescent-organelle reporter system in procyclic Trypanosoma brucei by expressing a fluorescent protein (FP) fused to a glycosomal targeting sequence (peroxisome-targeting sequence 2 [PTS2]). In these cell lines, PTS2-FP is localized within import-competent glycosomes, and organelle composition can be analyzed by microscopy and flow cytometry. Using this reporter system, we have characterized parasite populations that differ in their glycosome composition. In glucoserich medium, two parasite populations are observed; one population harbors glycosomes bearing the full repertoire of glycosome proteins, while the other parasite population contains glycosomes that lack the usual glycosome-resident proteins but do contain the glycosome membrane protein TbPEX11. Interestingly, these cells lack TbPEX13, a protein essential for the import of proteins into the glycosome. This bimodal distribution is lost in low-glucose medium. Furthermore, we have demonstrated that changes in environmental conditions trigger changes in glycosome protein composition. These findings demonstrate a level of procyclic glycosome diversity heretofore unappreciated and offer a system by which glycosome dynamics can be studied in live cells. This work adds to our growing understanding of how the regulation of glycosome composition relates to environmental sensing.T rypanosoma brucei, the causative agent of human African trypanosomiasis, has a complex life cycle, with developmental stages in the bloodstream of the mammalian host and the tsetse fly vector. Each host provides a distinct environment in which the parasites must survive. Bloodstream-form (BSF) parasites are bathed in glucose and generate ATP exclusively by glycolysis. While in the tsetse fly, the procyclic-form (PF) parasites experience a drop in glucose levels with a concomitant increase in the availability of amino acids (namely, proline). Under these conditions, the parasite adapts its metabolism, generating ATP from both glycolysis and amino acid metabolism (1).In trypanosomes, many of the enzymes involved in glycolysis are contained within membrane-bounded organelles called glycosomes (reviewed in references 2, 3, and 4). Similarities between the metabolic activities and the matrix protein import machineries of glycosomes and peroxisomes indicate an evolutionary relationship between the two organelles. In contrast to peroxisomes, however, glycosomes are essential, making mechanisms of glycosome biogenesis and maintenance attractive drug targets.Glycosome dynamics are governed by a number of processes, including organelle biogenesis, protein import, and changes in protein composition. In t...

Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum

“…), glycosome composition (Bauer et al. ) and the requirement for glycosome protein import in the PCF parasite (Furuya et al. ) is influenced by extracellular glucose levels.…”

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

“…Changes in glycosome composition can be monitored via following cellular fluorescence. Using this system, we have found that environmental conditions, notably glucose, regulate glycosome composition 12 .…”

“…The sequence encoding the fusion protein is cloned into pXS2bla 12 , which contains the procyclin promoter and the tubulin intergenic regions, which direct homologous recombination into the genome and the blasticidin resistance gene for selection. Procyclic cell lines harboring the genes encoding the T7 polymerase and tetracycline repressor (PF29-13) are transformed with the targeting construct, pXS2:PTS2eYFP.…”

Trypanosoma bruceiPex13.2 is an accessory peroxin that functions in the import of PTS2 proteins and localizes to subdomains of the glycosome

“…Cells were harvested (5×10 7 , 800 g 10 min), washed three times with PBS, and fixed (2% paraformaldehyde, 2.5% glutaraldehyde in 100mM phosphate buffer pH 7.4). Cells were stored at 4°C for no longer than 2 days before being processed as described previously ( 23 ). Glycosome area measurements were performed using FIJI.…”