The basic cell biology of African trypanosomes, which causes disease in both humans and cattle, has been understudied, particularly regarding protein trafficking and secretion. Therefore, the Specific Aims of this application are to: 1) To develop soluble secretory reporters for expression in trypanosomes by transient and stable transformation. These will then be exploited by genetic manipulation to investigate the nature of secretory protein targeting in Trypanosoma brucei. 2) To clone and characterize biochemical components of the T. brucei secretory machinery. Two candidate proteins have been selected for investigation, the trypanosomal homologues of ERD2 and hsc70. ERD2 is the membrane receptor that mediates retention of lumenal proteins in the ER. Identification of this polypeptide will provide a unique marker for the post- ER pre-Golgi compartment in trypanosomes further defining the secretory pathway in this organism. Hsc70 is a molecular chaperone involved in translocation of secretory polypeptides into the ER lumen and in vesicular trafficking. The goals of this proposal form a multifaceted, but integrated, approach to the processes of protein trafficking in trypanosomes. It is expected that the results of these studies will provide original insights into the nature of protein trafficking in trypanosomes, underscoring aspects of essential processes that are common to all eukaryotes, as well as reveling feature that are unique to trypanosomes.