Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

Abstract

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single‐celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity‐dependent biotin identification (BioID)‐based proteomics approach, using the established IMC marker protein Photosensitized INA‐Labelled protein 1 (PhIL1) as bait in asexual blood‐stage parasites. Subsequent mass spectrometry‐based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP‐tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood‐stage parasites.

Inner membrane complex proteomics reveals a palmitoylation cascade regulating intraerythrocytic development of malaria parasite

“…Exact localization of these proteins at the parasite pellicle need to be determined in the future using other methods, such as super resolution imaging, immunoelectron microscopy, or split green fluorescent protein. We noted that Wichers et al recently explored an IMC proteome in asexual blood stages of P. falciparum with BioID using PhIL1 as the bait [30]. Of the 225 PhIL1-interacting protein candidates, the orthologs of 37 proteins are listed among the 300 Tb-IMC interacting proteins in our study (Table S1).…”

“…Although the collection of 300 proteins may contain some false positives, two lines of evidence suggest good reliability of this IMC proteome. Firstly, about 50 IMC or IMC-associated proteins have been identified in the Plasmodium to date [5, 30]. Among these, 30 proteins (60%) have orthologs included in the Tb-IMC interacting proteins (Table S1).…”

“…BioID has been used to identify the protein components of complexes and organelles in different model organisms [24]. In the Plasmodium , BioID-based PL had also generated organelle- and vesicle-specific proteins or proteomes, including those of the gametocyte-specific osmiophilic bodies of P. berghei , the blood stage parasitophorous vacuolar membrane of P. berghei and P. falciparum , the apicoplast of P. falciparum , and the IMC and apical annuli proteins of P. falciparum [25–30]. These analyses have led to the functional discovery of previously undescribed proteins, providing new insights in the organelle biology of malaria parasites.…”

PMRT1, aPlasmodiumspecific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

“…For co-localization experiments the plasmids pLyn-FRB-mCherry (37), P40PX-mCherry (40), pARL- crt ACP-mCherry (42), pARL- ama1 ARO-mCherry (44) and pARL- ama1 AMA1-mCherry (47) were used. For conditional gametocyte induction yDHODH was amplified by PCR from pARL- ama1 AMA1-mCherry-yDHODH (47) and cloned into GDV1-GFP-DD-hDHFR(56)(56) using the XhoI/XhoI restriction site. For expression in Xenopus oocysts Pf PMRT1 was amplified from c- nmdr Pf PMRT1-ty1 and DNA fragments were ligated and inserted into the oocyte expression vector pGEM-HE (76), containing a C-terminal eYFP using the BamHI/XhoI restriction site.…”

Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

“…For overexpression constructs the full length PfApiAT10 sequence was amplified from parasite gDNA and cloned into pARL-ama1 46 -AIP-mCherry-yDHODH 61 using the XhoI/KpnI restriction site or into the pARL-crt 45 -PF3D7_0324600-GFP-hDHFR 37 plasmid using the KpnI/AvrII restriction site. Oligonucleotides used to generate the DNA fragments are summarized in Table S1.…”