Figure 1. Deletion of the Rem C-terminus prevents plasma membrane localization.

(A) Diagram showing features of the Rem C-terminus and the locations of Rem truncations. (B) TsA201 cells were transfected with plasmids expressing Rem^WT and either empty pCMVT7F2 vector, Ca_V1.2 and/or Flag-Ca_Vβ2a. 72 hours post-transfection, cells were examined by confocal microscopy. The localization of Rem^WT at the cell periphery is not significantly affected by co-expression of calcium channel components. (C) TsA201 cells were transfected with plasmids expressing Rem truncations and either empty pCMVT7F2 vector, Ca_V1.2 and/or Flag-Ca_Vβ2a, as described in Figure 1B. GFP-Rem^1-265 and GFP-Rem^1-270 show cytosolic localization, GFP-Rem^1-276 shows slight cell periphery enrichment, and GFP-Rem^1-282 displays very strong cell periphery enrichment consistent with plasma membrane localization irrespective of Ca_Vβ2a or Ca_V1.2 co-transfection. (D) Confocal images were quantified by line-scan from the cytosolic interior of the cell to the plasma membrane as described under “Experimental Procedures”. Intensity at the cell periphery was divided by the mean intensity over the total line-scan to find cell peripheral enrichment. Line-scan was performed four times for each cell examined and the results averaged. A significant difference (p<0.05) between treatments is denoted by asterisks. (E) Selection of tsA201 cells from Figures 1C and 1D. Arrows indicate patches of increased GFP-Rem^1-276 expression at the cell boundary.