Protease Trafficking in Two Primitive Eukaryotes Is Mediated by a Prodomain Protein Motif

Abstract

Trypanosome protozoa, an early lineage of eukaryotic cells, have proteases homologous to mammalian lysosomal cathepsins, but the precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent protein as a reporter, we demonstrate that the carbohydrate-free prodomain of a trypanosome cathepsin L is necessary and sufficient for directing green fluorescent protein to the lysosome/endosome compartment. A proper prodomain/catalytic domain processing site sequence is also required to free the mature protease for delivery to the lysosome/endosome compartment. A nine-amino acid prodomain loop motif, implicated in prodomain-receptor interactions in mammalian cells, is conserved in the protozoa. Site-directed mutagenesis now confirms the importance of this loop to protease trafficking and suggests that a protein motif targeting signal for lysosomal proteases arose early in eukaryotic cell evolution.Sorting of newly translated lysosomal proteases may occur by two different mechanisms. In mammalian cells, the predominant is through the addition of phosphomannosyl residues and targeting to the lysosome pathway by binding to M6P 1 receptors within the Golgi (1). However, transport of lysosomal enzymes in many cells is unaffected by a deficiency in the phosphotransferase, which is required for M6P synthesis (2). M6P-receptor-independent membrane association has been reported for several lysosomal proteins (3). Confirmation and further analysis of a M6P-independent sorting pathway in mammalian cells has been complicated by both the presence of the M6P pathway itself and difficulty in distinguishing effects on protein folding versus protein trafficking when deletional mutants of the protease precursors were analyzed (4, 5). Trypanosoma cruzi and Leishmania mexicana are protozoa (Trypanosoma) representing one of the earliest lineages of eukaryotic cells. They nevertheless express proteases homologous to cathepsins L and B and, unlike yeast, have an organelle ultrastructure more reminiscent of mammalian cells (6). While the cathepsin L-like protease prodomains of these organisms have significant homology to mammalian procathepsin L (e.g. 45% identity for cruzain versus mouse cathepsin L), there are no carbohydrate modifications, so they are unique experimental models for analyzing M6P-independent protein sorting.Trypanosome cysteine proteases are synthesized as precursor proteins with a hydrophobic signal peptide, a 100 -122-amino acid prodomain, a 200 -210-amino acid catalytic domain, and, in most cases, a 100 -130-amino acid carboxyl-terminal domain (7-12). The function of the carboxyl-terminal domain is as yet unknown. Hypotheses that it plays a role in protease inactivation, or in facilitating folding of the catalytic domain, have been ruled out by expression of fully active recombinant proteases without this domain (10). Other proposed functions have included mediating intracellular trafficking of the protease (13), immune evasion (14), and facilitating activity against specific macromolecular substrates (15).The pr...

Glycosome turnover inLeishmania majoris mediated by autophagy

“…Therefore a similar analysis of vesicle size was performed in stationary phase promastigotes expressing GFP-ATG8 and proCPB-RFP, which is known to label the lysosome. 4 This more extensive analysis produced similar results, with GFP-ATG8-labeled vesicles exhibiting a mean diameter of 0.36 mm, whereas vesicles labeled with both GFP-ATG8 and proCPB-RFP, or proCPB-RFP alone, had mean diameters of 0.59 mm and 0.53 mm, respectively (Fig. 6B).…”

“…To determine whether this compartment was the lysosome, we employed 2 different markers: FM 4-64 6,21 and proCPB-RFP, the propeptide region of the lysosomal cysteine peptidase B. 4 Both of these markers colocalized with GFP-ATG8 in large structures similar to those colabeled by GFP-ATG8 and RFP-SQL (Fig. 5C), showing that this compartment is the lysosome.…”

“…3 These remodeling events involve increased protein turnover, as evidenced by the evolution of lysosome morphology and associated increases in expression of peptidases. 3 The lysosomal compartment occurs as a single large vesicular structure at the anterior end of procyclic promastigotes, 4,5 whereas in the metacyclic promastigote it is tubular and known as the multivesicular tubule (MVT) or MVT-lysosome. 5,6 Intracellular amastigotes possess characteristic large lysosomal compartments known as megasomes, which vary in size and number depending on the Leishmania species.…”

The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

“…Toxopain-1 does not appear to have mannose 6-P despite two potential glycosylation sites in its prodomain (data not shown). McKerrow and coworkers (40) has identified a prodomain motif in the cathepsin Ls of T. cruzi and Leishmania mexicana, which are required for targeting to lysosomes, but similar signals have not been identified in cathepsin B. We also found that toxopain-1 is released extracellularly into buffer independent of synchronized rhoptry release during invasion.…”

Falcipain Cysteine Proteases Require Bipartite Motifs for Trafficking to the Plasmodium falciparum Food Vacuole

“…These positively charged amino acids might be crucial for the structural stability of the prodomain and/or its interactions with proteins required for proper trafficking. Similarly, positively charged residues play a key role in trafficking the cysteine protease cruzain to the lysosome/endosome compartment in the protozoan Trypanosoma cruzi (28). On the cytoplasmic side of the falcipain-2 prodomain, a Phe-Val motif between residues 15 and 25 is conserved across homologues of falcipains (Fig.…”