Biochemical Fractionation of Trypanosomes for the Analysis of Glycosomal Protein Import Defects.

Insect-transmitted trypanosomatid parasite infections cause life-threatening neglected tropical diseases (NTDs), including African sleeping sickness, Chagas disease and leishmaniasis. In these parasites, glycosomes are unique organelles that are essential for the parasite survival. Proper biogenesis of glycosomes is crucial to ensure correct compartmentation of the glycosomal metabolism. Genetic or chemical disruption of the glycosome biogenesis leads to a mislocalization of the glycosomal enzymes into the cytosol, which results in toxicity to the parasites. Here, we describe a detailed protocol for biochemical fractionation of Trypanosoma brucei parasites to detect mislocalization of glycosomal proteins to the cytosol. This approach utilizes increasing concentrations of digitonin that first permeabilizes the plasma membrane, followed by permeabilization of other organelles, depending on their cholesterol content. Fractionated samples can be further analyzed using immunoblotting for specific marker proteins or quantified by the specific enzyme activities.