Research ArticleSIGNAL TRANSDUCTION

The CCT chaperonin is a novel regulator of Ca2+ signaling through modulation of Orai1 trafficking

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Science Advances  26 Sep 2018:
Vol. 4, no. 9, eaau1935
DOI: 10.1126/sciadv.aau1935
  • Fig. 1 The C-terminal cytoplasmic domain of Orai1 is required for its targeting to the PM.

    (A) Cartoon representation of YFP-HA-Orai1 and Orai1 deletions: Orai1-ΔN(90-301), Orai1-ΔC(1-256), and C-terminal Orai1 truncations (1-266, 1-275, and 1-285). (B) Western blot probed with anti-HA antibodies from cells transfected with YFP-HA-Orai1 (WT), Orai1-ΔC, or Orai1-ΔN with β-actin as a loading control. (C) Representative images from CHO cells transfected with YFP-HA-Orai1, Orai1-ΔC, or Orai1-ΔN and probed with Cy3-anti-HA to label exofacial HA epitopes. Scale bar, 5 μm. (D) Quantification of S/T Orai1 for the full-length Orai1, ΔN, and ΔC. Data are the means ± SEM of 3 to 28 experiments depending on the construct. Surface levels of ΔN and full-length Orai1 were equivalent, whereas Orai1-ΔC showed significantly decreased (“*”, P = 0.0177) surface levels. (E) CHO cells transfected with WT Orai1, Orai1-ΔC, 1-266, 1-275, or 1-285 were fixed without permeabilization and stained with α-HA antibody followed by a Cy3-labeled secondary antibody. S/T Orai1 levels for the different deletions were determined by the Cy3/YFP ratio and normalized to WT. Data are the means ± SEM of three independent experiments. “***” indicates statistically significant datasets (P < 0.0057) as compared to WT. (F) Time course of cell-associated α-HA as a measure of exocytosis of the different Orai1 C-terminal deletions. Data were fitted to an exponential growth function to determine the exocytosis rate. The data are the means ± SEM of three independent experiments. (G) Estimates of Orai1 percentage that recycles at the PM (see text for details).

  • Fig. 2 Proteomic analysis of Orai1 interactomes.

    (A) Anti-HA antibody feeding enriches Orai1 sub-PM vesicles. Representative images 2 and 45 min after α-HA feeding. The Cy5 signals show surface Orai1, whereas the Cy3 signal labels intracellular Orai1. (B) Western blot probed with anti-Orai1 of different fractions from a sucrose gradient to enrich for intracellular endogenous Orai1. Probing for the PM marker Na-K ATPase shows no PM contamination. Gel lanes of interest were entirely excised after Coomassie staining, digested with trypsin, and analyzed by MS. (C) Immunoprecipitation approach to pulldown Orai1-positive vesicles. (D) Summary of MS analysis for Orai1 interactome from the immunoprecipitation-based approach and subcellular fractionation–based approach. Av H/L, average heavy/light.

  • Fig. 3 CCT complex binds to expressed and endogenous Orai1 and regulates Orai1 levels at the PM.

    (A) YFP-HA-Orai1 complexes were immunoprecipitated from stably (S) or transiently (T) transfected YFP-HA-Orai1 CHO cells using GFP microbeads, separated on SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and probed with specific antibodies against CCT2, CCT1, CCT5, and HA. (B) Coimmunoprecipitation (IP) of endogenous Orai1-CCT complexes. CHO cell lysates input (Inp.) incubated with antibodies against CCT2, CCT1, or CCT5 as the immunoprecipitating antibody or control IgG followed by Western blotting using Orai1 antibody. The middle blot shows efficient CCT2 pulldown using the CCT2 antibody. The blot on the right shows that the CCT1, CCT2, and CCT5 coimmunoprecipitate. (C) Reciprocal immunoprecipitation with antibody against Orai1 followed by Western blotting using CCT2, CCT1, and CCT5 antibodies. Some lysates were expressing CCT2-GFP. (D) Knockdown of endogenous CCT2 by siRNA. Cell lysates were separated on SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with CCT2 and actin antibodies, followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG secondary antibodies. Quantitative Western blot analysis was performed using LI-COR. (E) Stable YFP-HA-Orai1 CHO cells treated with CCT2 siRNA (50 nM) or nontargeting siRNA (control) for 48 hours and stained with anti-HA antibodies followed by a Cy5-labeled secondary. The surface Orai1–to–total Orai1 ratio [(Cy5/YFP in nonpermeabilized cells)/(Cy5/YFP in permeabilized cells)] was determined by quantitative immunofluorescence as indicated in Materials and Methods. Data are the means ± SEM from three independent experiments. **P < 0.01.

  • Fig. 4 Identifying CCT binding domain in Orai1.

    (A) Coimmunoprecipitation of CCT from CHO cells expressing WT, Orai1-ΔC, and Orai1-ΔN, using GFP microbeads. Lysates from cells transfected with a pEGFP-C1 (GFP) were used as control. (B) Sequence alignment of the Lox-1 receptor cytoplasmic domain shown to bind CCT with the Orai1-ICL. The scrambled ICL construct (ICL-S) is shown in red. (C) Representative epifluorescent images of a mutant Orai1 bearing the scrambled sequence 157NEKPHRSLVES167 (ICL-S). Scale bar, 5 μm. (D) Orai1 was immunoprecipitated from cells expressing WT or ICL-S mutant and probed with CCT2 antibody. Binding of CCT2 to ICL-S Orai1 was determined by the ratio of bound CCT2 to total immunoprecipitated Orai1 normalized to WT. (E) Surface Orai1–to–total Orai1 ratio in CHO cells expressing either WT or ICL-S Orai1. (F) Time-dependent uptake of anti-HA antibody in CHO cells expressing either WT or ICL-S Orai1 as a measure of exocytosis rate. Data were fitted with a monoexponential growth curve to determine the exocytosis rate constant. (G) Time course of intracellular accumulation of HA bound Orai1 measured as internal (Cy3/YFP) over surface (Cy5/YFP). The data were fitted by a linear regression. The endocytosis rate constant was calculated using the following relation: Orai1surface × Kendo = Orai1Internal × Kexo. The data are the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 CCT-Orai1 interaction modulates Ca2+ signaling downstream of Orai1.

    (A) Ca2+ transients in response to 50 μM ATP stimulation in Ca2+-containing (2 mM) Ringer solution measured using Fura-2 in CHO cells cotransfected with STIM1 and Orai1 (red) or STIM1 and ICL-S Orai1 (blue). Untransfected cells were used as control (black). Time to 75% decay was determined (means ± SEM from three independent experiments). (B) Accelerated STIM1-Orai1 puncta formation in CHO and HeLa cells transfected with ICL-S Orai1 after TG-dependent calcium store depletion. Live-cell images by TIRF microscopy and intensity recordings showing accumulation of the subplasmalemmal puncta in response to 1 μM TG in CHO cells cotransfected with STIM1 and Orai1 (red) or STIM1 and ICL-S (blue). (C) Time course for NFAT1 nuclear translocation in response to store depletion with TG (1 μM) (top). Cells were cotransfected with CFP (cyan fluorescent protein)–STIM1, YFP-Orai1, and GFP-NFAT1 or with CFP-STIM1, ICL-S, and GFP-NFAT1. Ratio of GFP-NFAT fluorescence in the nucleus/cytosol over time. The initial slope of N/C NFAT was calculated within 200 s after TG (bottom). *P < 0.05, **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaau1935/DC1

    Supplementary Methods

    Fig. S1. Effects of Orai1 C-terminal truncations on SOCE.

    Fig. S2. Subcellular localization of Orai.

    Fig. S3. Orai1 PM targeting is independent of its expression levels and glycosylation.

    Fig. S4. Replacing residues 157 to 167 in the Orai1-ICL with alanine prevents Orai1 trafficking to the PM.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • Fig. S1. Effects of Orai1 C-terminal truncations on SOCE.
    • Fig. S2. Subcellular localization of Orai.
    • Fig. S3. Orai1 PM targeting is independent of its expression levels and glycosylation.
    • Fig. S4. Replacing residues 157 to 167 in the Orai1-ICL with alanine prevents Orai1 trafficking to the PM.

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