Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Abstract

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1: Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFPLdgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.Small GTPases of the Rab family are master regulators of intracellular trafficking (1). These proteins are localized on specific compartments and regulate the transport through fusion between two compartments in a nucleotide-dependent process (2, 3). About 70 Rab proteins are identified in mammalian cells. In the endocytic pathway, Rab5 is present in the sorting endosome, whereas Rab4 and Rab11 are localized in recycling endosomes (4, 5). Rab7, Rab9, and Rab24 are associated with the late endosomal compartment. Rab7 mediates transport from the late endosomes to lysosomes, whereas Rab9 regulates the trafficking of lysosomal enzymes from the trans-Golgi network to lysosomes (6 -9). In the secretory pathway, Rab1 regulates the anterograde transport from the endoplasmic reticulum (ER) 3 to the Golgi (10), whereas retrograde transport from Golgi to ER is mediated by Rab2 (11). Rab6 localizes in the Golgi and is involved in intra-Golgi trafficking (12). Moreover, Rab18, Rab30, and Rab43 are found to be important for maintaining Golgi structure (13,14). In addition, transport of cargo from the Golgi to the cell surface is regulated by different Rabs. For example, Rab3 plays a role in release of neurotransmitter, Rab27 regulates the trafficking of lytic granules and melanosomes toward the plasma membrane, and Rab8 is involved in regulating the traffic from the trans-Golgi network to the plasma membrane (15). However, the secretory pathway is n...

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

“…However, how Ldgp63 exits from ER and follows the secretory pathway in the parasites is not known. 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).…”

“…To understand the functions of Leishmania Sar1 more precisely, two mutants viz, GTP-locked (H74L) and GDP-locked (T34N) were generated by PCR mediated site directed mutagenesis using appropriately designed mutant primers as described previously (36). Briefly, megaprimer for H74L was generated using WT Sar1 forward primer and mutated reverse primer (5 / -GCCTGCTGAAGGCCACCCATATC-3 / ) by PCR and subsequently full length mutated product was generated using this megaprimer as forward primer with WT reverse primer.…”

“…Respective cells containing indicated construct were grown separately in LB and induced with 0.5 mM isopropyl 1-thio-Dgalactopyranoside for 3 h at 37 °C for the expression and purification of respective GST or His 6 fusion proteins using reduced glutathione or Ni-NTA beads, respectively, by standard procedures (36) .…”

“…GTP binding activity of purified LdSar1:WT and its mutants was detected by GTP overlay assay (36). Briefly, 2 µg of GSTLdSar1:WT or its mutants was blotted onto nitrocellulose membrane and membrane was incubated with 1 µCi/ml of [α-…”

“…The GTP hydrolysis activities of LdSar1:WT and its mutants were determined as described previously (36). Briefly, 5 µg of immobilized protein on glutathione beads was incubated with buffer A (20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 5 mM MgCl 2 , 1 mM NaH 2 PO 4 and 10 mM β-mercaptoethanol) for 20 min at 25⁰C and bound nucleotide was eluted with 1 M guanidine-HCl.…”