PfNT2, a Permease of the Equilibrative Nucleoside Transporter Family in the Endoplasmic Reticulum of Plasmodium falciparum

Abstract

The survival and proliferation of the obligate intracellular malaria parasite Plasmodium falciparum require salvage of essential purines from the host. Genetic studies have previously shown that the parasite plasma membrane purine permease, PfNT1, plays an essential function in the transport of all naturally occurring purine nucleosides and nucleobases across the parasite plasma membrane. Here, we describe an intracellular permease, PfNT2. PfNT2 is, like PfNT1, a member of the equilibrative nucleoside transporter family. Confocal and immunoelectron microscopic analyses of transgenic parasites harboring green fluorescent protein-or hemagglutinin-tagged PfNT2 demonstrated endoplasmic reticulum localization. This localization was confirmed by colocalization with the endoplasmic reticulum marker PfBiP. Using yeast as a surrogate system, we show that targeting PfNT2 to the plasma membrane of fui1⌬ cells lacking the plasma membrane nucleoside transporter Fui1 confers sensitivity to the toxic nucleoside analog 5-fluorouridine. This study provides the first evidence of an intracellular purine permease in apicomplexan parasites and suggests a novel biological function for the parasite endoplasmic reticulum during malaria infection.

Nutrient Transport and Pathogenesis in Selected Parasitic Protozoa

“…Upon completion of the malaria genome project (39), 3 more NT genes (PfNT2 to PfNT4) were discovered, but the transport properties of the corresponding permeases are largely unknown, with the possible exception of the endoplasmic reticulum-resident PfNT2, which may transport uridine (34). To determine the role of PfNT1 in intraerythrocytic parasites, Ben El Bissati and coworkers (36) disrupted the PfNT1 gene using a double-crossover strategy.…”

Identification and Functional Analysis of the Primary Pantothenate Transporter, PfPAT, of the Human Malaria Parasite Plasmodium falciparum

“…The cells were fixed, washed, depleted of their cell wall, and permeabilized, and immunofluoresence analyses were performed as previously described (5). Localization of PfPAT-GFP and native PfPAT (using PfPAT antibody) in P. falciparuminfected erythrocytes by immunofluorescence and immunoelectron microscopy were carried out as previously described (5,29).…”

“…These clones were not further pursued. The inability to identify the pantothenate transporter using this library could be due to either the absence of the plasmid harboring the transporter cDNA in the library or the inability of the transporter to localize to the yeast plasma membrane (5). Therefore, we searched the library of putative P. falciparum metabolite/drug transporters for possible homologs of eukaryotic and prokaryotic pantothenate and vitamin permeases.…”

“…studies have shown that, more often than not, expression and characterization of P. falciparum transporter genes in the yeast S. cerevisiae requires codon optimization of the gene of interest, reduction of the overall A ϩ T base content, and amelioration of the distribution of codon usage frequency along the length of the gene sequence (5,28,35,36). Therefore, for successful expression of PfPAT, the codon usage and GC content of this gene were adjusted to produce PfPAT co .…”

“…The uptake of these essential nutrients involves endogenous transporters at the erythrocyte membrane as well as parasite-encoded permeases targeted to red blood cell membrane, parasite plasma membrane, or intracellular organelles (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The water-soluble vitamin pantothenic acid (vitamin B 5 ) is a precursor of the important enzyme cofactor CoA, a universal carrier of activated acyl groups involved in 9% of biochemical reactions identified in all living organisms (15). Because P. falciparum cannot synthesize pantothenate de novo, the acquisition of this vitamin from the host is an indispensable nutritional function for the parasite (Fig.…”