Receptor-mediated endocytosis in the procyclic form of Trypanosoma brucei

In Trypanosomatids, endocytosis and exocytosis occur exclusively at the fla gellar pocket, a deep invagination of the plasma membrane where the flagell um extends from the cell. Both bloodstream and procyclic trypanosomes are c apable of internalizing macromolecules, However, structures resembling coat ed vesicles were only identified in bloodstream form and not in procyclic f orm trypanosomes. Due to the apparent absence of coated vesicles in procycl ics, the significance of receptor-mediated endocytosis in procyclic trypano somes has been considered of minimal importance. We show that the flagellar pocket associated cysteine-rich acidic transmembrane protein (CRAM) may fu nction as an high density lipoprotein receptor in the procyclic form trypan osome. Using anti-CRAM IgG we have characterized the process of CRAM-mediat ed endocytosis in procyclic form trypanosomes, The wild type procyclic tryp anosome binds and internalizes anti-CRAM IgG but not the non-immune IgG in a saturable and time-dependent manner; the binding and uptake of I-125-labe led anti-CRAM IgG are inhibited by excess unlabeled anti-CRAM IgG. Uptake a nd degradation of anti-CRAM IgG do not occur at 4 degrees C, At 28 degrees C, the internalized anti-CRAM IgG were efficiently degraded through a proce ss that is inhibited by incubation at 4 degrees C and sensitive to the pres ence of chloroquine. The uptake and degradation of anti-CRAM IgG does not o ccur in the CRAM null mutant cell line, These results suggested that the up take of anti-CRAM IgG in the wild type procyclics occurs via receptor-media ted endocytosis of the CRAM protein. Deletion of the cytoplasmic extension of CRAM drastically reduced the degradation but not the binding of anti-CRA M IgG, This result indicated that potential internalization signals may be present in the cytoplasmic extension of CRAM. This is the first time that t he importance of receptor-mediated endocytosis in procyclic form trypanosom es has been demonstrated.