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

Abstract

Metalloprotease 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 LdSec241-702 to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec311-603, and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine594/595 and valine597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24703-966, 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.