Interacts with Orai1 (93). The interaction calls for an 110aa192349239 | PNAS | November 29, 2011 | vol. 108 | no.CAuthor contributions: G.K. and D.E.C. designed analysis; G.K., L.K., S.C.S., and Y.M. performed analysis; G.K., L.K., and Y.M. contributed new reagents/analytic tools; G.K., S.C.S., and D.E.C. analyzed information; and G.K. and D.E.C. wrote the paper. The authors declare no conflict of interest.To whom correspondence really should be addressed. E mail: [email protected]. edu.This short article contains supporting facts on-line at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1117231108//DCSupplemental.www.pnas.org/cgi/doi/10.1073/pnas.Fig. 1. POST binds Orai1 and is expressed inside the ER and plasma membrane. (A) HA epitopetagged POST especially binds myctagged Orai1 coexpressed in HEK 293 cells. Cell lysates were immunoprecipitated with HAagarose and stained on Western blot (WB) with myc or HAantibody conjugated to HRP. (B) Endogenous POST binds Orai1. Jurkat cell lysates have been immunoprecipitated with Orai1, POST antibody, or preimmune rabbit IgG, and probed on WB with all the indicated antibody (TrueBlot). Lysates of HEK 293 expressing mycOrai1 (labeled as mycOr1) and HAPOST have been utilized as markers. Note that retailer depletion (cells treated with 1 M thapsigargin for ten min in Ca2free Ringer’s remedy) didn’t affect POSTOrai1 interaction. (C) Live confocal fluorescence pictures of HEK 293 cells coexpressing POSTGFP and CherrySTIM1. POST was localized in the cell periphery and intracellular membrane network, where it precisely colocalized with Cherry STIM1 (ER). Note the fraction of POST that is expressed within the cell periphery, exactly where STIM1 is not expressed.antiPOST antibodies have been generated in rabbits. These antibodies especially immunoprecipitated their target proteins and identified Orai1 [35 and 42 kDa bands (glycosylated), 32.six kDa predicted] and POST (35kDa band, 39.8 kDa predicted) on Western blots (Fig. 1B and Fig. S3) but weren’t suitable for immunofluorescent staining of native proteins. Immunoprecipitation (IP) of endogenous Jurkat Orai1 and POST confirmed that these proteins are components of a molecular complex and revealed that POSTOrai1 binding did not depend on ER Ca2 content material (Fig. 1B).On Retailer Depletion, POST Binds STIM1 and Moves for the Plasma Membrane. GFPPOST expressed in HEK 293 cells localized to anaction. IP of endogenous POST from Jurkat cells recovered the STIM1 OST molecular complex only below storedepleted circumstances (Fig. 3B).POST, Like STIM1, Can also be Present within the Plasma Membrane. STIM1 was initially identified as a plasma membrane protein, but most STIM1 is found within the ER. Because we originally identified POST binding for the endogenous plasma membrane channel, Orai1, we wondered no matter whether POST may also be present on the plasma membrane. Indeed, simultaneous TIRF microscopy of GFPPOST and CherrySTIM1 in cells with full Ca2 stores revealed that POST may very well be observed in thin appendages lacking CherrySTIM1 (Figs. 1C and 2C and Film S1). Lastly, surface biotinylation of HEK 293 proteins clearly demonstrated the presence of endogenous transmembrane POST Loracarbef Biological Activity protein inside the plasma membrane (Fig. S5). Quantification of biotinylation indicates that 50 of POST is positioned in the plasma membrane. Thus, like STIM1, POST is both an ER protein in addition to a plasma membrane protein. POST Overexpression or Downregulation Doesn’t Substantially Affect Calcium Entry through Orai1. POST binding to Orai1, also asintracellular membrane network, whe.