| Giardia lamblia is a eukaryotic protozoan parasite and the causative agent of giardiasis, a debilitating enteric disease resulting in much morbidity and mortality worldwide. It is of interest not only as a target for the development of improved giardiasis therapies, but also as a model eukaryotic system. Giardia represents the earliest branching clade of eukaryotic cells. It is thus an ideal system for investigating the evolution of cell processes, organelle compartmentalization, and critical protein families. Analysis of the structure and function of the Giardia endomembrane system, cysteine proteases, and clathrin orthologues are the focus of this dissertation project.; The ER has been studied recently as a putative endocytic organelle. Examination of Giardia endocytosis using fluorophore-labeled proteins revealed that proteins were rapidly trafficked to a tubulovesicular network with ER-like properties. Using reporter constructs, cysteine proteases that are orthologues of lysosomal hydrolases were localized to the same tubulovesicular network. Functional protease assays helped define the role that cysteine proteases play in the degradation of exogenous proteins. Organelle-specific markers were used to describe the tubulovesicular compartment in which endocytosis and subsequent proteolysis takes place.; Cysteine proteases have been implicated in life cycle transitions (encystation and excystation) of Giardia. The completion of the Giardia genome indicated that there are twenty-seven cysteine protease genes in Giardia. Cysteine protease 2 (GlCP2) was identified as the major expressed cysteine protease gene in Giardia . Biochemical analysis of heterologously expressed GlCP2 suggested that this gene indeed plays an important role in both encystation and excystation.; Giardia clathrin is also key to the processes of endocytosis and encystation. Clathrin is associated with the peripheral vacuoles of vegetative Giardia and may facilitate the initial endocytic uptake of proteins. During encystation, clathrin localizes to encystation specific vesicles and may function in cyst formation. A dominant negative clathrin heavy chain disrupted cyst formation but did not affect endocytosis. |
