Putative lysosomal targeting motifs were detected using the Protein Motif Pattern tool (https://trichdb.org/trichdb/app/search/transcript/GenesByMotifSearch). M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the Telithromycin (Ketek) introduction of glycosylation sites to secreted -amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases. beta-sandwich repeat protein; TCA, trichloracetic acid; TEAB, triethylammonium bicarbonate; TGN, trans Golgi network; TLCK, tosyl-L-lysyl-chloromethane hydrochloride; TMD, transmembrane domain; TSP, tetraspanin; TYM, tryptone-yeast extract-maltose medium; UCE, uncovering enzyme, N-acetylglucosamine-1-phosphodiester -N-acetylglucosaminidase; vATPase, vacuolar ATPase Graphical Abstract Open in a separate window is a flagellated parasitic protist that causes the most common nonviral Telithromycin (Ketek) sexually transmitted disease, with 276 million new infections annually (1). In women, the parasite can cause vaginitis and increase the risk of HIV transmission, preterm delivery, low birth weight, and cervical cancer. Most infected men are asymptomatic, but long-term infections increase the risk of prostate cancer development (2, 3, 4, 5). In the vaginal mucosa, parasites actively phagocytose host cells such as epithelial cells, lymphocytes, erythrocytes, as well as cell debris and microbes, including yeast and bacteria (6, 7, 8, 9). Moreover, secretes a large number of biologically active molecules, such as adhesins for cytoadherence, cytotoxic cysteine proteases (CPs), amylases and glycosidases to metabolize available glycogen (4, 10), and peptidoglycan hydrolases that are active against the bacterial cell wall (11). secretes proteins through the classical secretory pathway (10) or unconventionally exosomes, which are derived from the endolysosomal pathway (12). Vesicles with engulfed material fuse with lysosomes to form phagolysosomes, which are acidic organelles specializing in the breakdown of a broad range of biomolecules (13, 14, 15, 16). In addition, lysosomes participate in various other cellular processes, such Telithromycin (Ketek) as autophagy (17, 18), secretion, and degradation of misfolded proteins within the secretory pathway (19). The biogenesis of lysosomes depends on the delivery of newly synthesized proteins from the trans-Golgi network (TGN) the transport vesicles that deliver cargo within the cell and through the endosomal pathway that imports proteins from the plasma membrane (20, 21). Lysosomes are supplied with over 60 hydrolases (15, 22), as well as other proteins such as acidifying vacuolar ATPases (vATPases), lysosome-associated membrane glycoproteins (LAMPs), and over 50 lysosomal channel proteins and transporters (14, 15). In metazoans, the sorting of most lysosomal hydrolases depends on the mannose 6-phosphate (M6P) pathway (23). Soluble lysosomal proteins are glycosylated on asparagine residues within the sequence Asn-X-[Ser/Thr] (soluble lysosomal targeting sequence, s-LTS) in the endoplasmic reticulum (ER) (23, 24). Then, in the Golgi body, N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) and N-acetylglucosamine-1-phosphodiester -N-acetylglucosaminidase (uncovering enzyme, UCE) form M6P for interaction with M6P receptors (MPRs). Two MPRs, a cation-dependent (CD-MPR) and cation-independent MPR (CI-MPR), have been identified (22, EIF4EBP1 23, 25). Other lysosomal sorting receptors have also been described, including LIMP-2 (mammals), VSR (plants, algae, alveolates), and sortilin/Vps10, which were studied in mammals and yeast; however, sortilin homologues have been identified in members of all eukaryotic groups (23). Novel receptors for the delivery of CPs to lysosomes (CP-binding protein family 1) were identified in (26). Receptors involved in protein delivery to lysosomes consist of a luminal domain that binds cargo in the Golgi body, at least one transmembrane domain, and a C-terminal tail that contains a lysosomal targeting sequence (LTS) facing the cytosol. LTSs of transmembrane proteins and receptors (t-LTS) are most frequently dileucine-based ([DE]xxxL[LI], DxxLL) or tyrosine-based (Yxx?) (24) and regulate endosomal/lysosomal sorting and internalization from the plasma membrane (27). T-LTSs are bound by cytosolic Golgi-localized, -ear-containing, ADP ribosylation factor-binding proteins (GGAs) that mediate sorting at the TGN, which is further facilitated by adaptor proteins. Cargo is released into acidified endosomes, and receptors are Telithromycin (Ketek) recycled. However, GGAs.