Hemoglobin endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket

Four lines of evidence indicate that a specific high affinity binding site on the surface of Leishmania donovani promastigotes mediates rapid internal ization and degradation of hemoglobin. 1) Binding and uptake of I-125-hemog lobin by Leishmania followed saturation kinetics and were competed by unlab eled hemoglobin but not by globin or hemin or other heme- or iron-containin g proteins. 2) Immunogold labeling studies revealed that, at 4 degrees C, h emoglobin binding was localized in the flagellar pocket of the promastigote s. Indirect immuno-fluorescence assays showed that, at 37 degrees C, the bo und hemoglobin in such cells entered an endocytic compartment within 2 min and dispersed throughout the cell body by 15 min. 3) After incubation with hemoglobin-gold conjugates at 25 degrees C or 37 degrees C, the particles a ccumulated in discrete intracellular vesicles. 4) A single biotinylated pro tein of 46-kDa was revealed when solubilized membranes from surface biotiny lated intact Leishmania adsorbed by hemoglobin-agarose beads were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with avidin -horseradish peroxidase, Considered together, these data indicate that this 46-kDa protein on the cell surface of L, donovani promastigotes mediates t he binding of hemoglobin and its rapid internalization through a vesicular pathway characteristic of receptor-mediated endocytosis.