Fig. 4.

Rab7 induced the transport of Hb to the late compartment. (A) To determine the role of Rab7 in Hb transport to the lysosomes in Leishmania, B-Hb transport to avidin-HRP preloaded lysosomes was measured in Rab7, and its mutants overexpressed cells as described in Materials and Methods. At respective time points, cells were lysed and B- Hb-avidin-HRP complexes were immunoprecipated from the lysate by anti-Hb antibody. HRP activity associated with the complex was measured as relative transport to the lysosomes. Transport observed at 60 min in untransfected control cells was taken as 100%, and results are expressed as relative percentage of transport to the lysosomes from three independent experiments ± SD. One hundred percent transport in untreated control cells corresponds to 15.9 ng of HRP activity per milligram of protein. (B and C) Uptake (B) and degradation (C) of ¹²⁵I-Hb in LdRab7 or its mutant overexpressed Leishmania. Uptake and degradation of ¹²⁵I-Hb by LdRab7 or its mutant overexpressed Leishmania along with untransfected control cells were determined as described in Materials and Methods. At respective times, cell-associated radioactivity (B) and trichloroacetic acid-soluble noniodide radioactive products (C) released in the medium were determined. Results are expressed as nanograms of Hb per milligram of cell protein from three independent determinations ± SD.