The broad goal of this proposal is to understand basic processes of secretory protein transport in African trypanosomes, with special regard to glycosylphosphatidylinositol (GPI) membrane anchors. These processes have been traditionally studied in yeast and mammals, but the availability of sophisticated genetic strategies in Trypanosoma brucei provides a potent alternative system to study eukaryotic secretory cell biology. Importantly, two features make African trypanosomes especially relevant for study. First, trypanosomes are high impact human and veterinary pathogens in sub-Saharan Africa. The WHO estimates that >60 million people in 36 countries are at risk of acquiring Human African Trypanosomiasis (HAT). Few drugs are available, the best of which (eflornithine) is expensive and requires a difficult regimen, the worst of which (melarsoprol) kills up to 10% of recipients. Infection is always fatal without intervention, vaccination is not an option, and there is a critical need for new therapies. Second, the ancient phylogenetic status of trypanosomes, and their unique secretory architecture, ensure that novel results relative to the `standard' eukaryotic model systems will be obtained. The lynchpins of pathogenesis in trypanosomiasis are the GPI- anchored variant surface glycoprotein (VSG), and related transferrin receptor (TfR), of the bloodstream stage of the life cycle. Understanding how they are transported to the cell surface, and the role of GPIs in this trafficking, are critical to understanding the parasite half of the host-parasite relationship. This proposal is designed to investigate these processes. The Specific Aims all derive directly from progress from the previous funding period. First, we have identified a gene family of p24 membrane proteins as putative loading receptors for exit of secretory cargo, including VSG, from the ER (Aim #1). Second, we have demonstrated that non- functional GPI-anchored proteins are monitored and degraded by the ERAD (ER-Associated Degradation) pathway (Aim #2). Third, we have developed a strategy for in situ modification of the TfR heterodimer subunits (ESAG6/ESAG7) that allows us to test the hypothesis that GPI valence regulates progression/stability of proteins within post-Golgi pathways (Aim #3). These results will drive deeper investigations of secretory trafficking in trypanosomes to illuminate critical aspects of basic trypanosome cell biology. It is our belief that this work may help lay the foundation for future drug development, and will highlight not just the differences, but also the similarities of cell biological processes common to the full range of eukaryotic evolution.