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

J Biol Chem. 2017 Jul 21;292(29):12111-12125. doi: 10.1074/jbc.M117.784033. Epub 2017 Jun 2.

Abstract

Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63L594A/L595A or GFP-Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion.

Keywords: GTPase; Leishmania; Sar1; Secretory pathway; exocytosis; gp63; protein secretion; trafficking.

MeSH terms

  • Amino Acid Substitution
  • COP-Coated Vesicles / enzymology*
  • COP-Coated Vesicles / metabolism
  • Cell Line, Tumor
  • Cytosol / enzymology
  • Cytosol / metabolism
  • GTP Phosphohydrolases / chemistry
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism*
  • Humans
  • Intracellular Membranes / enzymology
  • Intracellular Membranes / metabolism
  • Leishmania donovani / cytology
  • Leishmania donovani / genetics
  • Leishmania donovani / growth & development
  • Leishmania donovani / metabolism*
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Macrophages / cytology
  • Macrophages / metabolism
  • Macrophages / parasitology*
  • Metalloendopeptidases / chemistry
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism*
  • Mutagenesis, Site-Directed
  • Mutation
  • Organisms, Genetically Modified
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Interaction Domains and Motifs
  • Protein Transport
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Vesicular Transport Proteins / chemistry
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism
  • Virulence Factors / chemistry
  • Virulence Factors / genetics
  • Virulence Factors / metabolism*

Substances

  • Luminescent Proteins
  • Peptide Fragments
  • Protozoan Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Vesicular Transport Proteins
  • Virulence Factors
  • Metalloendopeptidases
  • glycoprotein gp63, Leishmania
  • GTP Phosphohydrolases