A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum

Marchetti, Rosa V.; Lehane, Adele M.; Shafik, Sarah H.; Winterberg, Markus; Martin, R. L.; Kirk, Kiaran

doi:10.1038/ncomms7721

Cited by 59 publications

(83 citation statements)

References 40 publications

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“…(A) Subfamily logo and averaged specific amino acid side‐chain volumes at the proposed selectivity filter, and the lipophilic constriction sites. The subfamily logo was calculated from a representative set of 41 eukaryotic and 30 prokaryotic FNT protein sequences and depicts subfamily discriminating residues weighted by the information content. (B) Top views of the putative selectivity filters of prokaryotic FocA (left, narrow diameter fitting a formate substrate molecule) and eukaryotic Pf FNT (right, wider diameter).…”

Section: Resultsmentioning

confidence: 99%

A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

et al. 2017

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Bacterial formate-nitrite transporters (FNT) regulate the metabolic flow of small weak mono-acids derived from anaerobic mixed-acid fermentation, such as formate, and further transport nitrite and hydrosulfide. The eukaryotic Plasmodium falciparumFNT is vital for the malaria parasite by its ability to release the larger l-lactate substrate as the metabolic end product of anaerobic glycolysis in symport with protons preventing cytosolic acidification. However, the molecular basis for substrate discrimination by FNTs has remained unclear. Here, we identified a size-selective FNT substrate filter region around an invariant lysine at the bottom of the periplasmic/extracellular vestibule. The selectivity filter is reminiscent of the aromatic/arginine constriction of aquaporin water and solute channels regarding composition, location in the protein, and the size-selection principle. Bioinformatics support an adaptation of the eukaryotic FNT selectivity filter to accommodate larger physiologically relevant substrates. Mutations that affect the diameter at the filter site predictably modulated substrate selectivity. The shape of the vestibule immediately above the filter region further affects selectivity. This study indicates that eukaryotic FNTs evolved to transport larger mono-acid substrates, especially l-lactic acid as a product of energy metabolism.

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Section: Resultsmentioning

confidence: 99%

A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

et al. 2017

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“…In each case, the transport properties of the recombinant protein closely resembled the characteristics of a transport process detected in the parasite. That said, whilst the expression of PfFNT in Xenopus oocytes (Marchetti et al ., ) and yeast (Wu et al ., ) produced somewhat similar transport profiles, the kinetics of monocarboxylate transport across the parasite's plasma membrane (Elliott, Saliba & Kirk, ) most closely matched the transport properties of PfFNT when the protein was expressed in the oocyte system (Marchetti et al ., ). This observation further underscores the potential for an expression system to affect the apparent activity and function of the recombinant transporter.…”

Section: The Plasmodium Transportomementioning

confidence: 99%

“…These determinations have both increased in throughput and become more economical with the introduction of a 96-well format (Richards et al, 2016) and the development of a fluorescence-based assay for measuring the transport of substrates (van Schalkwyk et al, 2016). Thus far, 19 Plasmodium transporters have been expressed and characterised in this system, including PfHT1 (Woodrow et al, 1999;Woodrow, Burchmore & Krishna, 2000), PfENT1 (Carter et al, 2000;Parker et al, 2000), PfENT4 (Frame et al, 2012), the P i :Na + symporter PfPiT (Saliba et al, 2006), PfAQP (Hansen et al, 2002;Promeneur et al, 2007), PfCRT Summers et al, 2014;Richards et al, 2016;van Schalkwyk et al, 2016), the formate-lactate channel PfFNT (Marchetti et al, 2015;Hapuarachchi et al, 2017), PfCAX (Rotmann et al, 2010), the cationic amino acid transporter PbNPT1 (Rajendran et al, 2017), and PfHLYIII (Moonah et al, 2014). Nevertheless, not all attempts to express Plasmodium transporters in the Xenopus oocyte have been successful (Henry et al, 2007;Cobbold, Llinas & Kirk, 2016) and in a few cases the signal-to-background ratio was very modest, such that it prevented direct and robust measurements of the transporter's activity [e.g.…”

Section: (4) Known or Predicted Transport Functionsmentioning

confidence: 99%

The transportome of the malaria parasite

Martin

2019

Biological Reviews

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Membrane transport proteins, also known as transporters, control the movement of ions, nutrients, metabolites, and waste products across the membranes of a cell and are central to its biology. Proteins of this type also serve as drug targets and are key players in the phenomenon of drug resistance. The malaria parasite has a relatively reduced transportome, with only approximately 2.5% of its genes encoding transporters. Even so, assigning functions and physiological roles to these proteins, and ascertaining their contributions to drug action and drug resistance, has been very challenging. This review presents a detailed critique and synthesis of the disruption phenotypes, protein subcellular localisations, protein functions (observed or predicted), and links to antimalarial drug resistance for each of the parasite's transporter genes. The breadth and depth of the gene disruption data are particularly impressive, with at least one phenotype determined in the parasite's asexual blood stage for each transporter gene, and multiple phenotypes available for 76% of the genes. Analysis of the curated data set revealed there to be relatively little redundancy in the Plasmodium transportome; almost two‐thirds of the parasite's transporter genes are essential or required for normal growth in the asexual blood stage of the parasite, and this proportion increased to 78% when the disruption phenotypes available for the other parasite life stages were included in the analysis. These observations, together with the finding that 22% of the transportome is implicated in the parasite's resistance to existing antimalarials and/or drugs within the development pipeline, indicate that transporters are likely to serve, or are already serving, as drug targets. Integration of the different biological and bioinformatic data sets also enabled the selection of candidates for transport processes known to be essential for parasite survival, but for which the underlying proteins have thus far remained undiscovered. These include potential transporters of pantothenate, isoleucine, or isopentenyl diphosphate, as well as putative anion‐selective channels that may serve as the pore component of the parasite's ‘new permeation pathways’. Other novel insights into the parasite's biology included the identification of transporters for the potential development of antimalarial treatments, transmission‐blocking drugs, prophylactics, and genetically attenuated vaccines. The syntheses presented herein set a foundation for elucidating the functions and physiological roles of key members of the Plasmodium transportome and, ultimately, to explore and realise their potential as therapeutic targets.

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“…This mechanism is complementary to that of ammonium transporters, e.g., E. coli AmtB, which are thought to abstract a proton from the ammonium cation, NH 4 C , for neutralization and subsequent passage of neutral ammonia, NH 3 , via a hydrophobic transport path. 8 Weak acid and base neutralizing transporters, thus, make use of the same mechanistic principle.…”

mentioning

confidence: 99%

“…On the cellular level, FNTs regulate the metabolic substrate flow and fulfill vital functions, for instance the Plasmodium falciparum PfFNT that we and others identified as the malaria parasite's long-sought lactic acid transporter. 3,4 Blockade of PfFNT by small druglike molecules kills the parasites rendering PfFNT a novel antimalarial drug target. 5,6 Their physiologic relevance as well as structural peculiarities merit a deeper look into the inner workings of the FNTs.…”

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confidence: 99%

Formate-nitrite transporters: Monoacids ride the dielectric slide

Wiechert

Beitz

2017

Channels

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The discovery that the Escherichia coli focA gene product facilitates the transmembrane exchange of formate 1 sparked research on proteins that turned out to be functionally situated between classical transporters and channels. 2,3 The strictly microbial protein family was named formate-nitrite transporters (FNT) according to the physiologic substrates of the 2 initially characterized members. However, subsequently, the substrate spectrum was extended and comprises several weak monoacids up to the size of lactic acid. On the cellular level, FNTs regulate the metabolic substrate flow and fulfill vital functions, for instance the Plasmodium falciparum PfFNT that we and others identified as the malaria parasite's long-sought lactic acid transporter.3,4 Blockade of PfFNT by small druglike molecules kills the parasites rendering PfFNT a novel antimalarial drug target. 5,6 Their physiologic relevance as well as structural peculiarities merit a deeper look into the inner workings of the FNTs.High resolution crystal data revealed a common homopentameric structure.2 The protomers consist of 6 transmembrane spans with both termini at the cytoplasmic side. Surprisingly, FNTs almost perfectly mimic the fold of the aquaporin water and solute channels despite unrelated amino acid sequences. The substrate path through each FNT protomer harbors 2 hydrophobic constrictions, which sandwich the imidazole sidechain of a highly-conserved histidine. The transport mechanism, however, remained unclear regarding to the question whether the substrate anion, e.g. formate, is required to be neutralized by protonation to pass the lipophilic constriction sites.Initially, electrophysiology was used to determine substrate selectivity and pH dependent transport of FocA.2 These studies showed electrogenic, i.e. anion, transport at neutral pH and very low, i.e., channellike, substrate affinity in the high millimolar range. Further, a sudden loss of anion transport was observed when the buffer pH was slightly shifted toward acidic conditions suggesting a gating mechanism for which different conformations of the N-terminal domain were made responsible.2 Together, FocA seemed to behave as a pH-gated anion channel.Due to their weak acidity, e.g., pK a/formate 3.8 and pK a/acetate 4.8, the FNT substrates interconvert between the negatively charged anion and the protonated, neutral acid in a chemical equilibrium. To detect not only the electrogenic anion transport of FocA and PfFNT but additionally capture the transport of the neutral acid substrate species, we used radiolabeled substrates in our assays.3,7 Strikingly and contrary to the electrophysiology studies, we observed increasing transport rates with increasing acidity of the assay media. Apparently, neither FocA nor PfFNT exhibited closure or gating. Further, the alkalization of the media during transport indicated that protons are co-transported with the substrate and disruption of the transmembrane proton gradient revealed that transport is driven by a proton-motive force.7 Together, our da...

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A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum

Cited by 59 publications

References 40 publications

A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

A widened substrate selectivity filter of eukaryotic formate‐nitrite transporters enables high‐level lactate conductance

The transportome of the malaria parasite

Formate-nitrite transporters: Monoacids ride the dielectric slide

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