A privileged ER compartment for posttranslational heteromeric assembly of an ion channel

Figures

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  Fig. 1.. hERG1b is sequestered in intracellular puncta. (A) Confocal images of fixed and immunostained HeLa cells transfected with hERG1a or (B) hERG1b plasmids. (C) Quantification of the number of puncta per cell; data are mean ± SD, n = 36 cells per condition, analyzed with Student’s t test (****P < 0.0001). (D) Confocal live cell images of iPSC-CM transfected with 1a-GFP or (E) 1b-GFP. (F) Quantification of number of puncta per cell; data are mean ± SD, n = 18 cells for 1aGFP, 12 cells for 1bGFP, analyzed with Student’s t test (****P < 0.0001); scale bar, 10 μm in large image and 1 μm in the Inset.

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  Fig. 2.. hERG1b puncta are localized to ER. (A–C) STED microscopy of iPSC-CM transfected with mRNAs encoding hERG1bSNAP labeled with JFX554 (magenta) and ER marker Halo-KDEL labeled with JFX650 (cyan). (D) Quantification of colocalization events thresholded with three different distances between the center of hERG1b puncta and the surface of the ER; data are mean ± SD, n = 16 regions of interest (Methods). (E) Cartoon of ER localization of hERG1b puncta. The scale bar is 10 μm for (A) and 500 nm for (B and C).

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  Fig. 3.. hERG1b is sequestered in a privileged compartment. (A) Immunostaining of HeLa cells transfected with hERG1bHA or (B) hERG1a-Y611HHA. (C) quantification of the number of puncta per cell; data are mean ± SD, n = 33 for hERG1b, n = 26 for 1a-Y611H, analyzed with a Student t test (****P < 0.0001). (D) experimental design for the pulse–chase (en rule) experiment. (E) HeLa cells transfected with either hERG1aSNAP, 1bSNAP, 1a-Y611HSNAP, or 1b-Y271HSNAP imaged at various time points provided in (D) and sample images provided for 0 h and 8 h after labeling. (F) Quantification of normalized corrected total fluorescence intensity (CTCF) plotted across time; hERG1a-Y611H and hERG1b-Y611H data are fitted with single exponential decay; hERG1a, hERG1b data are plotted with connected lines; data are mean ± SEM, n = 35 to 45 cells per condition; data with individual data points are provided in SI Appendix, Fig. S4. (G) Quantification of the number of puncta per cell; data are mean ± SD, n = 31 for hERG1b, n = 32 for 1a-Y611H, analyzed with a Student t test (****P < 0.0001); Inset on the Upper Right of (E) shows the Halo-tag expression (yellow) used to identify the transfected cells. Scale bar, 10…

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  Fig. 4.. An ER retention motif promotes sequestration of hERG1b in ERSA. (A) Confocal images of HeLa cells expressing FLAG-tagged hERG1b alone, (B) hERG1b with N15XN mutation, and (C) coexpression of WT hERG1a and hERG1b. (D) Quantification of the number of puncta per cell; data are mean ± SD (n = 30 cells per condition) analyzed with a one-way ANOVA (****P < 0.0001); scale bar, 10 μm in large image and 1 μm in Inset.

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  Fig. 5.. Are hERG1b puncta are condensates? (A) Confocal images of live HeLa cells expressing hERG1b (magenta) stained with Potomac-Yellow dye (cyan) labeling membranes; scale bar, 10 μm in the large image and 2 μm in the Inset (i and ii). (B) Quantification of percentage of number of hERG1b puncta colocalizing within 250 nm with Potomac-labeled membranous puncta (n = 15 cells). (C) Confocal images of live HeLa cell expressing hERG1bGFP with photobleaching of individual puncta imaged before and after photobleaching; Scale bar, 5 µm in large image and 1 µm in Inset. (D) Quantification of fluorescence intensity of the photobleached puncta before and after photobleaching; data are mean ± SEM; n = 10 puncta from different cells. Data are from a representative experiment repeated three times. (E) GFP tagged hERG1b amino-terminal domain (1 to 64 residues) undergoes phase separation, with fluorescence recovery after photobleaching of individual puncta. (F) Quantification of normalized fluorescence intensity after photobleaching; data are mean ± SEM; n = 13 puncta from different cells in a representative experiment repeated three times. (G) Droplet-promoting regions in hERG1b identified by…

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  Fig. 6.. Sequestered hERG1b proteins can be rescued by newly expressed 1a. (A) Experimental design. (B) Mock-transfected cells showing hERG1b puncta. (C) 1a-transfected cells showing hERG1b signal in or near plasma membrane in live cell imaging. (D) Quantification of the number of puncta per cell. Data are mean ± SD, n = 35 to 42 cells per condition and analyzed with a Student’s t test (****P < 0.0001). Scale bar, 10 μm in large image and 1 μm in Inset.