Transmembrane Facilitation of Lactate/H+ Instead of Lactic Acid Is Not a Question of Semantics but of Cell Viability.

Transmembrane transport of monocarboxylates is conferred by structurally diverse membrane proteins. Here, we describe the pH dependence of lactic acid/lactate facilitation of an aquaporin (AQP9), a monocarboxylate transporter (MCT1, SLC16A1), and a formate-nitrite transporter (plasmodium falciparum FNT, PfFNT) in the equilibrium transport state. FNTs exhibit a channel-like structure mimicking the aquaporin-fold, yet act as secondary active transporters. We used radiolabeled lactate to monitor uptake via yeast-expressed AQP9, MCT1, and PfFNT for long enough time periods to reach the equilibrium state in which import and export rates are balanced. We confirmed that AQP9 behaved perfectly equilibrative for lactic acid, i.e., the neutral lactic acid molecule enters and passes the channel. MCT1, in turn, actively used the transmembrane proton gradient and acted as a lactate/H+ co-transporter. PfFNT behaved highly similar to the MCT in terms of transport properties, although it does not adhere to the classical alternating access transporter model. Instead, the FNT appears to use the proton gradient to neutralize the lactate anion in the protein's vestibule to generate lactic acid in a place that traverses the central hydrophobic transport path. In conclusion, we propose to include FNT-type proteins into a more generalized, function-based transporter definition.

Cell-Free and Yeast-Based Production of the Malarial Lactate Transporter, PfFNT, Delivers Comparable Yield and Protein Quality.

Cell-free protein production is an attractive alternative to cell-based expression. Rapid results, small-volume reactions, irrelevance of protein toxicity, flexibility, and openness of the system are strong points in favor of the cell-free system. However, the in vitro situation lacks the cellular quality control machinery comprising e.g., the translocon for inserting membrane proteins into lipid bilayers, and chaperon-assisted protein degradation pathways. Here, we compare yield and protein quality of the lactate transporter, PfFNT, from malaria parasites when produced in Pichia pastoris yeast, or in an Escherichia coli S30-extract-based cell-free system. Besides solubilization and correct folding, PfFNT requires oligomerization into homopentamers. We assessed PfFNT folding/oligomerization and function by transmission electron microscopy imaging, transport assays, and binding of small-molecule inhibitors. For the latter, we used chromatography of the PfFNT-inhibitor complex with dual-wavelength detection, and biolayer interferometry. Our data show, that PfFNT possesses an intrinsic capability for assuming the correct fold, oligomerization pattern, and functionality during in vitro translation. This competence depended on the detergent present in the cell-free reaction. The choice of detergent further affected purification and inhibitor binding. In conclusion, in the presence of a suitable detergent, cell-free systems are very well capable of producing high quality membrane proteins.