This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Giardia is an important model for cell biology because it represents the most basal of eukaryotic lineages based on sequence analysis of conserved genes. Giardia trophozoites lack many typical eukaryotic organelles such as mitochondria, peroxisomes, and protein trafficking compartments such as a classical Golgi apparatus and secretory granules. Instead, Giardia employs a simple endomembrane system to export proteins to distinct intracellular locations. We have discovered a novel protein compartment in Giardia that appears to serve as ER and endosome. Using reporter-constructs, confocal microscopy, and ultrastructural analysis, we have shown that Giardia cysteine proteases localize to a tubulovesicular network with an ER-like structure peripheral to the perinuclear membrane. Labeled proteins are rapidly endocytosed into this compartment and degraded by these Giardia cysteine proteases. It therefore appears that Giardia contains a transitional endomembranous structure that may pre-date compartmentalization of endocytic/lysosomal functions and the endoplasmic reticulum. Giardia cysteine proteases may function to break down endocytosed nutrients. However, it remains unclear which members of the Giardia cysteine protease family are responsible for this activity. Cysteine protease activity can be isolated and purified from Giardia lysate using FPLC. This lysate fraction can then be submitted for mass spectrometry analysis to determine the gene products responsible for this novel activity.
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