The N-terminal residue for permitting membrane penetration and then tested their effect on Piezo1-SERCA2 interaction. The linker-peptide, but not the scrambled-peptide, reduced the interaction among Piezo| DOI: 10.1038s41467-017-01712-z | www.nature.comnaturecommunicationsNATURE COMMUNICATIONS | eight:NATURE COMMUNICATIONS | DOI: 10.1038s41467-017-01712-zARTICLE1.5 1.0 0.five n.s. 0.aPiezo1-GFP VectorAnti-FlagGFPMergedbFluorescence intensity ratio (F568F488)cn.s. kDa 300 300 130 anti-GST (biotinylated) anti-GST anti-Flag anti–actindNormalized biotinylated Piezo1.five 1.0 0.five 0.0 n.s.Piezo1-GFP SERCAWhole-cell lysatePiezo1-GFPSERCA2 Piezo1-A2419Flag-GFPVector Piezo1-A2419Flag-GFPSERCAPiezo1-GFPVectorPiezo1-A2419Flag-GFP VectorPiezo1-A2419Flag-GFP SERCAePiezo1-GFPAnti-FlagGFPMergedfFluorescence intensity ratio (F568F488)2.0 1.5 1.0 0.5 0.n.s.gPiezo1-GSTFlagPiezo1-GST SERCA2-Flaganti-GST (biotinylated) kDa 300 anti-GSThNormalized biotinylated Piezo1.five 1.0 0.five 0.Piezo1-A2419Flag-GFP300 anti–actinWhole-cell lysatePiezo1-GSTPiezo1-GSTFlagn.s. n.s.Piezo1-GFP Piezo1-A2419Flag-GFP (2172181)10A-A2419Flag-GFP KKKK-AAAA-A2419Flag-GFPGSTPiezo1-GSTKKKK-AAAA A2419Flag-GFPFig. 3 Neither SERCA2 co-expression nor the linker-mutations affect the expression of Piezo1 in plasma membrane. a and e, Reside immunofluorescent staining on the extracellularly localized Flag-tag inserted following the residue A2419 in the Piezo1-GFP, 2172181(10A)-GFP, and KKKKAAAA-GFP fusion proteins from HEK293T cells Acetylcholine Inhibitors Related Products transfected together with the indicated constructs. The GFP photos have been taken as control for the expression from the fusion proteins. Scale bar, 5 m. b and f, Scatter plots from the fluorescence intensity ratio from the anti-Flag signal (F568) over GFP signal (F488). Each dot represents the ratio of F568F488 from an individual cell. One-way ANOVA with a number of comparison test. c and g, Western blots of the biotinylated or whole-cell lysate samples derived from HEK293T cells transfected with the indicated constructs. d and h, Scatter plots in the normalized biotinylated Piezo1 levels of cells transfected with the indicated constructs. Unpaired student’s t-test (d) or One-way ANOVA with numerous comparison test (h). Data shown as imply s.e. m. p 0.and SERCA2 (Fig. 2h, i), indicating that the linker-peptide and Piezo1 compete for SERCA2 interaction. Collectively, these data recommend that the linker area serves as a crucial binding site for SERCA2. The identification on the important interacting residues in Piezo1 ABMA site provides compelling evidence that SERCA2 may possibly directly bind to Piezo1. This differs from previously identified Piezo1 regulatory proteins such as polycystein-2 (PC-2) and stomatin-like protein-3 (STOML3), which appears to regulate Piezo function through indirectly altering the membrane curvature or stiffness346. We hence went on to test how SERCA2 interaction could regulate Piezo1. No impact of SERCA2 or the mutations on Piezo1 localization. We initially examined no matter whether the plasma membrane expression of Piezo1 is impacted by SERCA2 co-expression or mutating the linker area (Fig. 3a). We inserted a Flag tag just after A2419 locatedNATURE COMMUNICATIONS | 8:within the extracellular CED28 into the Piezo1-GFP, 2172181(10A)GFP and KKKKAAAA-GFP fusion constructs (Piezo1A2419Flag-GFP, 2172181(10A)-A2419Flag-GFP and KKKK AAAA-A2419Flag-GFP, respectively), and after that carried out live immunostaining on the Flag tag from HEK293T cells transfected using the constructs without the need of permeabilizing the membrane. The GFP ima.
