The ubiquitin-proteasome pathway plays an essential role in proteolysis during Trypanosoma cruzi remodeling

Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch i n the eukaryotic lineage. The 20S proteasome of Trypanosoma cruzi epimastig otes was identified as a high molecular weight complex (1400 kDa) with an A TP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same elect rophorectic migration pattern as yeast 26S proteasome in nondenaturating ge ls. About 30 proteins in a range of 25-110 kDa were detected in the purifie d T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPa ses from eukaryotic 26S proteasome and the T, cruzi 20S core specifically r ecognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of th e ubiquitin (Ub)-proteasome system in protein degradation during the time o f parasite remodeling. Protein turnover in trypomastigotes was proteasome a nd ATP-dependent and was enhanced during the transformation of the parasite s into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphol ogical changes that occur during transformation, cytoskeletal proteins asso ciated with the flagellum are targets of the ubiquitin-proteasome pathway.