Giardia lamblia: an unique model of protein transport to lysosome in eukaryotes

MARIA ROMINA RIVERO; HUGO DANIEL LUJAN; THEODORE NASH; MARIA CAROLINA TOUZ

Mendoza- Argentina

Congreso; VII Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2005

Sociedad Argentina de Protozoología

Giardia lamblia is a primitive eukaryotic cell that possesses an unique secretory system. In more evolved cells, lysosomal proteins are sorted from the plasma membrane or trans-Golgi network to endosomal/lysosomal compartments by inclusion into clathrin-coated vesicles. Lysosomal trafficking is achieved by specific cargo recognition molecules (Adaptor Proteins or APs) that recognize specific sequences (tyrosine and/or dileucine motifs) in the cytoplasmic tail of membrane proteins. Giardia possesses peripheral vesicles (PVs) located underneath the plasma membrane that comprise a single endosomal/lysosomal compartment. We previously demonstrated that Giardia has a tyrosine-based sorting system, which mediates the targeting of an encystation-specific cysteine protease to the PVs in a clathrin-adaptin dependent process. We now show that receptor-specific transport of molecules from the parasite surface to the PVs (endocytosis) involves AP2. By tetracycline-induced expression of the medium subunit (µ) of AP2 and immunofluorescence assays we were able to localize Giardia AP2 in small vesicles close to the plasma membrane. Immunoblotting confirmed the correct molecular weight of the µ2. A µ2 antisense strategy and uptake experiments using FITC expressed labeled dextran and LDL suggest that AP2 is involved in receptor-mediated endocytosis. On the other hand, we recently reported also the delivering of the soluble lysosomal hydrolase acid phosphatase (AcPh) to the PVs is AP1 dependent. However, the nature of the receptor that sort AhPh to de PVs remains elusive. By pull-down assays using a His-tagged AcPh we were able to purify the recombinant enzyme together with associated proteins. Analysis of the sequency of associated proteins by mass spectrometry showed that molecules that potentially interact with AcPh in vivo are homologues to proteins involved in lysosomal transport in higher cells, indicatting that Giardia possesses a mechanism of protein delivery to lysosomes similar to more evolved cells. It is increasingly clear that its secretory pathway can be considered a stripped-down version of the more complex machinery present in higher eukaryotes. Further studies regarding additional molecules involved in lysosomal sorting in Giardia provide new insight into the minimal machinery necessary for intracellular transport in higher eukaryotes.