Abstract
In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells.
Highlights
Trafficking of newly synthesized lysosomal soluble enzymes from the trans-Golgi network (TGN) to lysosomes in mammalian cells occurs indirectly, via the plasma membrane followed by endocytosis, or directly, via the endosomal system
Trophozoites transfected with the vector pTubAcPh-V5/H6pac were grown in culture medium, and subcellular distribution of acid phosphatase (AcPh)-V5/H6 was analyzed by immunofluorescence
We recently showed that transport along the vacuolar pathway requires clathrin and the adaptors AP1 or adaptor protein 2 (AP2), with AP1 being involved in the forward lysosomal protein trafficking to the peripheral vacuoles (PVs), while AP2 participates in endocytosis [56]
Summary
Trafficking of newly synthesized lysosomal soluble enzymes from the trans-Golgi network (TGN) to lysosomes in mammalian cells occurs indirectly, via the plasma membrane followed by endocytosis, or directly, via the endosomal system. In these cells, the mannose 6-phospate receptors (MPRs) bind with high affinity to phosphomannosyl residues attached to the ligand proteins, while their cytoplasmic tails contain motifs capable of recognizing components of the clathrin-coated vesicles that are directed to the endosome/lysosome pathway. While in the TGN, the MPRs and Vps10p bind soluble hydrolases, their cytoplasmic tail packages them into clathrincoated vesicles, aided by adaptor proteins. Proper adaptor binding will result in delivery of hydrolases to the late endosomes [2,6]
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