Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania

Abstract

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a: N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptormediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a ؉/؊ and LdRab5b ؉/؊ cells. LdRab5a ؉/؊ Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b ؉/؊ cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in Leishmania

“…Recently, we have shown that Ldgp63 is trafficked via Rab1 dependent conventional secretory pathway in Leishmania (36). Moreover, we have also shown that Leishmania has conserved trafficking pathways like higher eukaryotic cells (37)(38)(39)(40)(41)(42)(43)(44). Thus, it is tempting to speculate that Ldgp63 might exit from ER by Sar1-dependent COPII mediated process.…”

“…Subsequently, Leishmania promastigotes were transfected with respective constructs by electroporation as described previously (36,40). Finally, positive clones were selected in the presence of G418 antibiotic at an initial concentration of 10 µg/ml and thereafter stably maintained at antibiotic concentration of 50 µg/ml.…”

“…To identify the LdSar1-positive compartment, LdSar1:WT-GFP overexpressing Leishmania were labeled with markers for different intracellular compartments as described earlier (36,40). The flagellar pocket was labeled with FM4-64 (30 μM) for 5 min at 4⁰C.…”

“…, LdSec24 703-966 or LdSec13 His 6 -fusion protein was immunized in BALB/c mice to raise antibodies by a standard method as described previously (40). The specificity of the antibodies against the respective proteins was determined by Western blot analysis using the respective purified proteins.…”

Single GDP-dissociation Inhibitor Protein regulates endocytic and secretory pathways in Leishmania

“…Both endocytic and secretory trafficking pathways regulated by various Rab GTPases are well conserved in Trypanosomatid parasites1819. We have also characterized role of Rab5 and Rab7 in the endocytic pathway of Leishmania 202122 using hemoglobin endocytosis in the parasite23242526. Similarly, we have found that Rab1 regulates the conventional secretory pathway in Leishmania 27.…”