Phosphorylation-Dependent Interactome of Ryanodine Receptor Type 2 in the Heart

Figure 1. RyR2 affinity-purification coupled to mass spectrometry. (A). Schematic showing the major methodological steps. LC = liquid chromatography; MS/MS = tandem mass spectrometry (B). Western blot validation of the immunoprecipitation of RyR2 from WT, RyR2 S2814A, and S2814D mouse ventricles. (C). Coomassie staining of gel containing samples from the various immunoprecipitations. Each lane (except for the input lane) is cut into ten equal pieces for in-gel digestion and LC-MS/MS analysis in parallel.

Figure 2. Phosphorylation-dependent alterations in cardiac RyR2 interactome. (A) RyR2 interactome defined based on enrichment with RyR2 pull-down compared to IgG (anti-RyR2/IgG) and beads-only (anti-RyR2/Beads) pull-downs. Ratios for each identified protein were calculated based on label-free quantification (LFQ) intensities across samples which were run and analyzed in parallel. Dotted lines mark ratios of 1.5, above which there is considered enrichment with RyR2 pull-down. The bait protein RyR2 (red) is highly enriched in the pull-down. Blue marks proteins enriched in RyR2 pull-down with respect to both IgG and beads-only controls (22 proteins). Yellow marks proteins enriched in RyR2 pull-down with respect to either IgG (15 proteins) or beads-only control (42 proteins). (B) Cross-validation and prioritization of WT, S2814A, and S2814D RyR2 IPs using published (List 1) [8] and unpublished (Lists 2 [10] and 3) putative RyR2 interactomes. List 3 is the putative RyR2 interactome generated in this study (Table S1). Enrichment score calculation is described in the Results section. (C). Alterations in the RyR2 interactome based on the phosphorylation-mimic status at S2814. Ratios for each…

Figure 3. Validation of RyR2 interactors identified from mass spectrometry. Western blot of RyR2 immunoprecipitated (IP) lysates from wild-type (WT) mouse ventricles showing RyR2 co-immunoprecipitation (co-IP) with Cpt1b (A), Aifm1 (B), and Idh3b (C). Mouse IgG was used as a negative control for these co-IP experiments. Representative blots from 3 independent experiments.

Figure 4. RyR2 S2814 phosphorylation-dependent interaction with Aifm1 and Idh3b. Western blot of RyR2 immunoprecipitated (IP) from lysates of ventricles from wild-type (WT), S2814A (SA), and S2814D mice showing differential RyR2 co-immunoprecipitation (co-IP) with Aifm1 and Idh3b (A). Quantification of co-immunoprecipitated proteins normalized to immunoprecipitated RyR2 (relative to WT) showing significantly reduced RyR2/Aifm1 (B) and RyR2/Idh3b (C) interactions in S2814D mice compared to both WT and S2814A mice. * p < 0.05; ** p < 0.01.

Figure 5. Changes in the RyR2 interactome based on phosphorylation status of S2814. As representatives of other putative interactors, Aifm1 and Idh3b have decreased binding to RyR2 S2814D mimicking phosphorylated RyR2 at S2814 (“Weak” arrow) but increased or “Strong” binding to RyR2 S2814A mimicking dephosphorylated RyR2 at S2814.