Figure 2.

YAP and TAZ regulates leucine uptake via SLC7A5. (A) YAP and TAZ are essential for LAT1 expression. LAT1 is a hetero dimeric transporter complex composed of SLC7A5 and SLC3A2, which are linked via a disulfide bond. Cell lysates from different cell lines as depicted in the box to the right were prepared in either non-reducing buffer (no β-mercaptoethanol) or reducing buffer (containing β-mercaptoethanol). Note that both the SLC7A5 antibodies and the SLC3A2 antibodies detect the high molecular heterodimeric complex under non-reducing condition (left part). Under the reducing condition (right half), both SLC7A5 and SLC3A2 run at the expected molecular weights of individual monomers. Ectopic expression of YAP (sample #a) rescues protein expression of both SLC3A2 and SLC7A5 in Y/T dbKO cells. (B) YAP/TAZ knockout decreases leucine uptake. H³-leucine uptake assay was carried out in cell lines (denoted in the right panel of Figure 2A) and normalized to WT cells. Error bars are means ± SEM of triplicates from a representative experiment. (C) LATS1/2 regulate leucine uptake via SLC7A5. Stable cell lines (right panel in Figure 2A) were generated and uptake of H³-leucine is displayed as relative to WT cells. Error bars are means ± SEM of triplicates from a representative experiment. (D) YAP/TAZ do not affect the plasma membrane localization of SLC7A5. WT and Y/T db KO cells that both stably express V5 epitope tagged SLC7A5 were co-cultured. YAP/TAZ WT cells are marked by positive YAP signal and dbKO cells (no YAP signal) are highlighted with white arrows. Ectopic SLC7A5 is localized to the plasma membrane in both WT and YAP/TAZ dbKO cells. Scale bar, 10 μm. (E) Reintroduction of SLC7A5 rescues the expression of SLC3A2 in YAP/TAZ knockout cells. Western blots of cell lysates from different cell lines as depicted. Re-expression of V5-tagged SLC7A5 or the epithelial specific SLC7A7, which likewise complexes with SLC3A2, but is not endogenously expressed in 293A cells, rescues the protein expression of SLC3A2. See also Supplementary information, Figure S3B. (F) Reintroduction of SLC7A5 restores leucine uptake in YAP/TAZ knockout cells. H³-leucine uptake assay was carried out in the indicated cell lines. #1 and #2 denotes two independent Y/T dbKO clones. SLC7A5 was re-expressed in Y/T dbKO clone #1. Error bars are means ± SEM of triplicates from a representative experiment.