High-level cell-free production of the malarial lactate transporter PfFNT as a basis for crystallization trials and directional transport studies.

The malaria parasite Plasmodium falciparum relies on the function of channel and transport proteins for the uptake of nutrients and the release of metabolic waste products. Inhibition of vital transport processes is an unexploited means for developing novel antimalarial drugs. The recently discovered plasmodial lactate transporter, PfFNT, represents a promising new drug target since the parasite's energy generation by anaerobic glycolysis depends on the rapid secretion of lactate. Yet, membrane proteins, in particular those of malaria parasites, are notoriously difficult to produce and purify in the native, functional form hampering crystallization and biophysical studies. Here, we show synthesis of milligram quantities of correctly folded PfFNT in a cell-free system. Solubilized PfFNT maintained its oligomeric, largely SDS-resistant quaternary structure and appears suitable for setting up crystallization trials. After reconstitution into proteoliposomes, PfFNT was functional as a transporter for formate, acetate, and lactate as determined by a light-scattering assay. Analysis of the accessibility of a protease cleavage site at the N-terminus revealed an even outside-in orientation of the total proteoliposomal PfFNT population that may be due to membrane curvature restrictions. Contrary to previous studies using heterologous expression in cell systems with oppositely oriented PfFNT, the proteoliposomes eventually allow for biophysical transport studies in the native, physiological direction.

Identity of a Plasmodium lactate/H(+) symporter structurally unrelated to human transporters.

Maintenance of a high glycolytic flow rate is critical for the rapid growth and virulence of malarial parasites. The parasites release two moles of lactic acid per mole of glucose as the anaerobic end product. However, the molecular identity of the Plasmodium lactate transporter is unknown. Here we show that a member of the microbial formate-nitrite transporter family, PfFNT, acts as a lactate/proton symporter in Plasmodium falciparum. Besides L-lactate, PfFNT transports physiologically relevant D-lactate, as well as pyruvate, acetate and formate, and is inhibited by the antiplasmodial compounds phloretin, furosemide and cinnamate derivatives, but not by p-chloromercuribenzene sulfonate (pCMBS). Our data on PfFNT monocarboxylate transport are consistent with those obtained with living parasites. Moreover, PfFNT is the only transporter of the plasmodial glycolytic pathway for which structure information is available from crystals of homologous proteins, rendering it amenable to further evaluation as a novel antimalarial drug target.

Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.