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. Author manuscript; available in PMC: 2009 Sep 6.
Published in final edited form as: Cell. 2009 Feb 26;136(5):876–890. doi: 10.1016/j.cell.2009.02.014

Figure 5. CAD Binds to Both the N- and C-termini of Orai1.

Figure 5

(A) In this split ubiquitin assay,β-gal production and growth of transformants on plates lacking histidine indicate a strong interaction between CAD and the C-terminus of Orai1, a weaker interaction with the N-terminus, and lack of interaction with the II–III loop of Orai1. (B) CAD in HEK 293T cells co-immunoprecipitates with YFP-tagged N-terminal and C-terminal fragments of Orai1 but not with the II–III loop. (C) Split ubiquitin assays showing that CAD interacts with aa 48–91 of the Orai1 N-terminus; this appears to be due to binding to aa 68–91 rather than aa 48–70. (D) CAD co-immunoprecipitates with Orai1-NT(48–91) but only very weakly with the entire N-terminus of Orai1 (aa 1–91). (E) Whole-cell recordings in 20 mM Ca2+o from HEK 293 cells co-expressing truncated Orai1-GFP proteins and YFP-CAD. Deletion of the N- or C-terminus of Orai1 abrogates function, but a substantial level of ICRAC is generated by Orai1-ΔN73. (F) Summary of ICRAC measurements (current at −100 mV, 20 mM Ca2+o, normalized to cell capacitance) with the indicated constructs (4 cells each, mean ± sem).