Screening of metabolite transporters by C isotope substrate labeling.

The transportome of 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 C-labeled stable isotopes and detection by targeted LC-MS/MS as a screening tool for identifying metabolite transporters. We compare the transport assay's sensitivity, reproducibility, and accuracy in yeast transporter mutant cell lines and 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 C-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 oocytes, followed by an uptake assay with C labeled yeast extract. Uptake assays in oocytes showed higher reproducibility and accuracy. Although having lower accuracy, the results from 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