EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

Abstract

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single‐pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR–DOZI‐aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S‐transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore‐forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM.

Malaria parasite plasmepsins: More than just plain old degradative pepsins

“…A variant of the chaperone hand-off model is that PM V could be in a subregion of the ER with a direct route to the PTEX translocon at the PVM (110,132). Subregions of the PVM have recently been described, with each region hosting distinct secretory proteins (133,134). However, there is no information yet about whether these PVM subregions are fed by distinct regions of the ER.…”

Contacting domains segregate a lipid transporter from a solute transporter in the malarial host–parasite interface

“…Thus, a fluorescent label for the PV lumen was required that could be compared with the distribution of EXP2-mNG. To this end, mRuby3 was targeted to the PV lumen using the signal peptide of HSP101 (PV-mRuby3) 15,16 . To verify that PV-mRuby3 would serve as a genuine label of the PV lumen, correlative light/electron microscopy after cryo-thin sectioning was performed 17 .…”

“…6), indicating that the EXP2-containing domains form the less strongly connected region. Recently, EXP1, a PVM protein that colocalizes with EXP2 23,24 , has been shown as important for peripheral EXP2 localization around the parasite and function of EXP2 as a nutrient-permeable channel 16,24 . It remains to be seen how EXP1 is affecting EXP2 localization and function.…”

Crossing the Vacuolar Rubicon: Structural Insights into Effector Protein Trafficking in Apicomplexan Parasites

“…EXP2 also serves as a small molecule/solute-permeable vacuolar channel [ 31 ] and is functionally equivalent to Toxoplasma gondii dense granule proteins GRA17 and GRA23 [ 34 , 35 ]. Two vacuolar separate molecular pools of EXP2 might thus coexist: One consisting of EXP2 assembled in the PTEX complex and one pool of ‘free’ EXP2 (without PTEX150 and HSP101), possibly associated with another vacuolar membrane protein, exported protein-1 (EXP1) [ 36 ], essential to the maintenance of the vacuole ultra-structure and EXP2 organization and function [ 37 ]. If EXP2 also exists under other functional form(s), does it adopt the same structure(s) and oligomeric state as in the context of PTEX?…”