Processing and targeting of cathepsin L (TbCatL) to the lysosome in Trypanosoma brucei

Cathepsin 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 RNA interference (RNAi) cell line targeting the CTD/3′ untranslated region (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 indicating trans-auto-catalytic activation. X′ is formed with active endoplasmic reticulum (ER)-retained TbCatLΔ:MDDL, but not with TbCatLΔ:C150A, indicating stochastic generation in the ER by cis-auto-cleavage within the prodomain of newly synthesized P′. Modelling 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 1/turn 1 junction, and a separate turn (T3). We demonstrate 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.