A conserved guided entry of tail-anchored pathway is involved in the trafficking of a subset of membrane proteins in Plasmodium falciparum

Abstract

Tail-anchored (TA) proteins are defined by the absence of N-terminus signal sequence and the presence of a single transmembrane domain (TMD) proximal to their C-terminus. They play fundamental roles in cellular processes including vesicular trafficking, protein translocation and quality control. Some of the TA proteins are post-translationally integrated by the Guided Entry of TA (GET) pathway to the cellular membranes; with their N-terminus oriented towards the cytosol and C-terminus facing the organellar lumen. The TA repertoire and the GET machinery have been extensively characterized in the yeast and mammalian systems, however, they remain elusive in the human malaria parasite Plasmodium falciparum. In this study, we bioinformatically predicted a total of 63 TA proteins in the P. falciparum proteome and revealed the association of a subset with the P. falciparum homolog of Get3 (PfGet3). In addition, our proximity labelling studies either definitively identified or shortlisted the other eligible GET constituents, and our in vitro association studies validated associations between PfGet3 and the corresponding homologs of Get4 and Get2 in P. falciparum. Collectively, this study reveals the presence of proteins with hallmark TA signatures and the involvement of evolutionary conserved GET trafficking pathway for their targeted delivery within the parasite. Tail-anchored (TA) membrane proteins are known to play essential cellular functions in the eukaryotes. These proteins are trafficked to their respective destinations by post-translational translocation pathways that are evolutionarily conserved from yeast to human. However, they remain unidentified in the malaria parasite Plasmodium falciparum. We have used bioinformatic prediction algorithms in conjunction with functional validation studies to identify the candidate TA repertoire and some of the homologs of the trafficking machinery in P. falciparum. Initially, we predicted the presence of 63 putative TA proteins localized to distinct compartments within this parasite, including a few confirmed TA homologs in other eukaryotic systems. We then identified and characterized PfGet3 as a central component in the Guided-Entry of TA (GET) translocation machinery, and our bacterial co-expression and pulldown assays with two selected recombinant TA proteins, PfBOS1 and PfUSE1, showed co-association with PfGet3. We also identified PfGet2 and PfGet4 as the other two components of the GET machinery in P. falciparum using proximity biotinylation followed by mass spectrometry. Interestingly, we also found six TA proteins in the parasite enriched in this fraction. We further validated the direct interactions between a few TA candidates, PfGet4 and PfGet2 with PfGet3 using recombinant-based pulldown studies. In conclusion, this study classified a subset of membrane proteins with the TA nomenclature and implicated a previously unidentified GET pathway for their translocation in this apicomplexan parasite.