• Molecular mechanisms of Intracellular Protein Trafficking During Cyst Wall Formation in Giardia lamblia.

ELÍAS VE; QUIROGA R; LUJÁN HD

Mexico City, Mexico

Congreso; X Ibero-American Congress on Cell Biology; 2007

Giardia trophozoites undergo fundamental changes to survive outisde the intestine of their hosts by differentiating into infective cysts. Encystation entails the synthesis, processing, trasnport, secretion, and assembly of cyst wall materials into a protective extracellular cyst wall. We are interested in deciphering the molecular events leading to the formation of the cyst wall, with emphasis on the vesicular transport of cyst wall components. In Giardia there are three closely related proteins that localize within encystation-specigic secretory transport vesicles (ESVs) in encysting trophozoites and in the cyst wall of mature cysts. The cyst wall protein genes predict proteins of 26 (CWP1), 39 (CWP2) and 27 (CWP3) kDa, having approximately 60% identity in a 26kDa overlapping region. CWP2 differs from CWP1 and CWP3 by a 121 residue carboy terminal extension rich in basic amino acids. In this work we (a) determined the role of the alkaline extension of CWP2 in ESV formation, and (b) identified and characterized SNARE proteins involved in Giardia encystation. First, CWP constructs with or without the CWP2 basic tail were transfected into trophozoites and two types of mammalian cell lines, those with and without endogenous secretory granules. Our results demonstrated that the CWP2 basic tail is necessary but not sufficient to induce the biogenesis of ESVs in non-encysting trophozoites and that there must be a receptor for this tail in Giardia since no granules were generated by expression of these proteins in mammalian cells. After their formation, little is known about the molecular mechanisms involved in vesicular docking and fusion of ESVs in Giardia. In higher eukaryotes, the soluble N-ehthylmaleimide-sensitive factor attachment protein receptors (SNARE) of the vesicle-associated membrane protein (VAMP) and syntaxin proteins play essential roles in the processes through the formation of complexes between proteins present on donor and target membranes. We present results from a search of the Giardia genome for SNARE-enconding genes, the analysis of the subcellular localization of their products, and the functionality of the SNAREs identified in this parasite. Our results indicate that each Giardia SNARE localizes to a particular subcellular compartment and that some of them are involved in the regulated secretory pathway occuring during cyst wall formation. By antisense knock-down xpression, we also found that most SNAREs are essential for parasite viability, demonstrating the importance of the SNARE proteins in constituting a minimal machinery for vesicle fusion in early-branching eukaryotes.