RESUMEN

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.

Asunto(s)

Sistema Libre de Células , Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/metabolismo , Proteolípidos/metabolismo , Proteolípidos/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Monosacáridos/química , Cinética , Antiportadores/metabolismo , Fenómenos Electrofisiológicos , Simportadores

RESUMEN

The transportome of Saccharomyces cerevisiae comprises approximately 340 membrane-bound proteins, of which very few are well-characterized. Elucidating transporter proteins' function is essential not only for understanding central cellular processes in metabolite exchange with the external milieu but also for optimizing the production of value-added compounds in microbial cell factories. Here, we describe the application of 13C-labeled stable isotopes and detection by targeted LC-MS/MS as a screening tool for identifying Saccharomyces cerevisiae metabolite transporters. We compare the transport assay's sensitivity, reproducibility, and accuracy in yeast transporter mutant cell lines and Xenopus oocytes. As proof of principle, we analyzed the transport profiles of five yeast amino acid transporters. We first cultured yeast transporter deletion or overexpression mutants on uniformly labeled 13C-glucose and then screened their ability to facilitate the uptake or export of an unlabeled pool of amino acids. Individual transporters were further studied by heterologous expression in Xenopus oocytes, followed by an uptake assay with 13C labeled yeast extract. Uptake assays in Xenopus oocytes showed higher reproducibility and accuracy. Although having lower accuracy, the results from S. cerevisiae indicated the system's potential for initial high-throughput screening for native metabolite transporters. We partially confirmed previously reported substrates for all five amino acid transporters. In addition, we propose broader substrate specificity for two of the transporter proteins. The method presented here demonstrates the application of a comprehensive screening platform for the knowledge expansion of the transporter-substrate relationship for native metabolites in S. cerevisiae.