Processing and targeting of Cathepsin L (TbCatL) to the Lysosome in Trypanosoma brucei

AbstractCathepsin L (TbCatL) is an essential lysosomal thiol protease in African trypanosomes. TbCatL is synthesized as two precursor forms (P/X) that are activated to mature form (M) with the removal of the prodomain upon arrival in the lysosome. We examine TbCatL trafficking in a novel system: truncated TbCatL reporter without the C ‐terminal domain (CTD) (TbCatL∆) ectopically expressed in an RNAi cell‐line targeting the CTD/3’UTR of endogenous mRNA. TbCatL∆ is synthesized as P’/X’/M’ species, localizes to the lysosome, and rescues the lethal TbCatL RNAi phenotype. Inactive TbCatLΔ:C150A is only processed to M’ in the presence of endogenous TbCatL indicatingtrans‐auto‐catalysis activation. X’ is formed with active ER‐retained TbCatLΔ:MDDL, but not with TbCatLΔ:C150A, indicating stochastic generation in the ER bycis‐auto‐cleavage within the prodomain of newly synthesized P’. Modeling the TbCatL prodomain on the human CatL structure suggests three solvent accessible features that could contain post‐Golgi targeting signals: the N‐terminus, the Helix 1̸ Turn 1 junction, and a separate turn (T3). We dem onstrate that the critical motif for lysosomal targeting is an asparagine‐proline dipeptide in T3 that is strictly conserved in all Kinetoplastida. These findings show novel insights on the maturation of TbCatL, which is a critical virulence factor in mammalian infection.
Source: Cellular Microbiology - Category: Microbiology Authors: Tags: RESEARCH ARTICLE Source Type: research