The African trypanosome Trypanosoma brucei is the causative agent of sleeping sickness in humans and of related diseases in livestock. T.brucei lives in the bloodstream and survives by undergoing antigenic variation of its major lariant ce1l- Surface Glycoprotein (VSG) coat. T.brucei depends on hosts for nutrients for growth and development. The observation that macromolecules, such as low density lipoprotein particles and transferrin enter trypanosomes via receptor-mediated endocytosis at the flagellar pocket, encouraged us to identify conserved surface proteins of the flagellar pocket. The flagellar pocket of trypanosomes, representing -0.43 percent of the pellicle membrane, is a deep invagination of the plasma membrane where the flagellum extends from the cell. In this pocket, the integrity of the VSG coat is broken and the subpellicular microtubule sheath is absent. Thus, the flagellar pocket forms a structurally and functionally discrete surface domain and is the only site where receptor mediated endocytosis takes place in Trypanosomatids. Our research focuses on elucidating the function of the flagellar pocket and on determining molecular determinants of protein trafficking from and to the pocket. We focused on a cysteine Rich Acidic-repetitive transMembrane protein (CRAM) located at the flagellar pocket of T.brucei. We determined that: i) CRAM may function as a lipoprotein receptor or an essential catalytic component of lipoprotein particle uptake and degradation; ii) we have shown that the cytoplasmic domain of CRAM is essential for CRAM mediated endocytosis; and iii) we have identified a putative transport and a putative retention signal, essential for localization of CRAM to the flagellar pocket, in the cytoplasmic extension of CRAM. This proposal aims to: 1) Determine mechanisms involved in routing and retention of CRAM to the flagellar pocket; 2) Determine mechanisms involved in CRAM mediated endocytosis; and 3) Identify and characterize proteins recognizing the putative sorting and internalization signals of the cytoplasmic extension of CRAM. These functional studies of a conserved invariant surface protein localized at the flagellar pocket will give us valuable insights into: i) molecular mechanisms involved in protein trafficking; ii) endocytosis of macromolecules; and iii) molecular components of biogenesis of the pocket. This study will reveal unique aspects of host-parasite interaction and may have practical applications for parasite control.
