Localisation of a multicatalytic protease complex in Trypanosoma brucei brucei

Abstract

Trypanosoma brucei brucei are tsetse-transmitted, extracellular protozoan parasite that causes Nagana, a cattle disease similar to sleeping sickness caused in humans by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Proteases of these parasites have come under increasing scrutiny because of their importance in such aspects as disease pathogenesis and as potential drug targets. Recently, a multicatalytic protease complex, the proteasome, has also become of interest because of its potential involvement in cell-cycle development. The proteasome is the central protease of the non lysosomal ubiquitin dependent pathway of protein degradation. Here we report the subcellular localization of 20S proteasome of Trypanosoma brucei brucei, using subcellular fractionation and enzymatic assays. Homogenate prepared from bloodstream form of T. brucei was fractionated by differential centrifugation into nuclear [NU] fraction, a large-granule [LG] fraction, small-granule [SG] fraction, microsomal [MI] and soluble cytosolic [CY] fractions. Each of these subcellular fractions was enriched in a particular organelle. The identity of these organelles was evaluated by assaying for the activity of marker enzymes. The proteasome activity was measured in the fractions using Z-Gly-Gly-Leu-AMC as substrate, peptide aldehyde ZLeu-Leu-Leu-H as specific inhibitor. The enzymatic activity was detected mainly in the cytosolic fraction (6.7-fold enrichment relative to the whole homogenate), but also in the nuclear fraction (2.8-fold). Our findings indicate that the internal location of the trypanosomal proteasome may not be restricted to the cytosol and nuclei as has previously been demonstrated in the eukaryotic cells.

Key words: Trypanosoma brucei, 20S proteasome. subcellular localization