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

Abstract

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures – the food vacuole, the apicoplast, and the parasite plasma membrane – and showed essentiality of four out of the six membrane transporters during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis. Heterologous expression of wild-type and mutation constructs in Xenopus laevis oocytes indicated ion transport upon membrane hyperpolarization and a functional role of negatively charged amino acids protruding into the parasitophorous vacuole lumen. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development and provide the first functional characterization of PfPMRT1, an essential parasite membrane transporter.

Iron transport pathways in the human malaria parasitePlasmodium falciparumrevealed by RNA-sequencing

“…This function may be performed by PfCRT (35) and / or PfNRAMP (36), which were both upregulated under high vs. low-iron conditions in our RNA-seq analysis (log 2 FC = 0.26, P = 0.007 and log 2 FC = 0.28, P = 0.003, respectively, Fig. 2B, Table 1) and are essential in asexual parasites (33,34,64). The predicted structure of PfNRAMP (Fig.…”

“…Based on transcriptomic profiles and data from the literature, six proteins with a potential role in iron transport were identified (Table 1 and 2). Of these, PfCRT (60) and PfNRAMP (34) had already been shown to localize to the digestive vacuole (DV) in live parasites (61). The subcellular localization of the other four putative iron transporters (PfMRS3, PfVIT, PfZIPCO, and PfE140) was then examined by endogenous tagging with GFP and confocal imaging of live parasites under physiological control conditions.…”

“…For instance, the vacuolar iron transporter PfVIT (PF3D7_1223700), an ortholog of Arabidopsis thaliana VIT1 (expect value (E) = 5 x 10 -29 , 30.5% identity, 87% coverage, as determined by position-specific iterated BLAST (29)), is a Fe 2+ /H + exchanger with unknown subcellular localization that likely plays a role in iron detoxification (30)(31)(32). The chloroquine resistance transporter PfCRT (PF3D7_0709000) and the natural resistance-associated macrophage protein PfNRAMP (PF3D7_0523800) were both localized to the digestive vacuolar (DV) membrane (33,34), and have been suggested to export Fe 2+ into the cytosol in symport with protons. These predicted functions are based on transport assays with Xenopus oocytes (35) and homology to the human endosomal Fe 2+ transporter 2/DMT1 (36), respectively.…”

“…Similarly, the Zrt-, Irtlike protein domain-containing protein (ZIPCO) was suggested to import Fe 2+ and Zn 2+ into the cytosol and localized to the parasite plasma membrane (PPM) in P. berghei sporozoites in indirect immunofluorescence assays (34), but PfZIPCO (PF3D7_1022300) had not been studied yet. The chloroquine resistance transporter PfCRT (PF3D7_0709000) and the natural resistance-associated macrophage protein PfNRAMP (PF3D7_0523800, also called PfDMT1 for divalent metal transporter 1, although this abbreviation is already in use for the drug/metabolite transporter 1) have been detected at the digestive vacuolar (DV) membrane (35,36). Both proteins were proposed to export Fe 2+ into the cytosol in symport with protons on the basis of transport assays using Xenopus oocytes (37) and proliferation assays with a conditional knockdown line under different iron conditions (38), respectively.…”