Identification of Novel Ryanodine Receptor 1 (RyR1) Protein Interaction with Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)*♦

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca2+ release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1–4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca2+ release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.

In skeletal muscle excitation-contraction (EC) 4 coupling is prominent among the processes controlled by Ca 2ϩ (1). The sequence of EC coupling events begins with the depolarization of sarcolemmal and transverse tubular membranes that triggers a conformational change in the dihydropyridine receptor (DHPR). Protein-protein interactions between the DHPR and the ryanodine receptor type 1 (RyR1) lead to the activation of the Ca 2ϩ release channel function of RyR1, the release of Ca 2ϩ from the SR via RyR1, and the generation of skeletal muscle contraction. Subsequently, relaxation is initiated by re-uptake of intracellular Ca 2ϩ through the action of the sarco/endoplasmic reticulum Ca 2ϩ -ATPase type 1 (SERCA1a). The correct functioning of the contraction/relaxation cycle relies on a precise balance between Ca 2ϩ release and re-uptake. Disruptions in this balance, such as those that result from mutations in the proteins involved in the Ca 2ϩ uptake and release processes, lead to muscle diseases, including Brody disease, malignant hyperthermia, central core disease, and cardiomyopathies (2)(3)(4)(5).
RyR1 has numerous interacting protein partners; that is, both integral membrane proteins such as DHPR, junction, and triadin (6) and soluble proteins such as FK506-binding proteins, calmodulin, protein kinases, and phosphatases (6 -9). These protein interactors are proposed to comprise an RyR1 Ca 2ϩ release channel complex and to regulate RyR1 function at the molecular level. Previous studies elucidating RyR1 structure and topology showed that RyR1 can be subdivided into a soluble, N-terminal, cytosolic domain (amino acids 1-4243) and an insoluble, C-terminal, pore-forming sector (amino acids 4244 -5038) containing at least 6 transmembrane sequences and a cytosolic tail (10).
In this study we attached codons for a 6ϫ-histidine affinity tag to the 3Ј end of rabbit cDNA encoding amino acids 1-4243 of RyR1. The soluble, C-terminal-tagged cytosolic domain (cytosolic RyR1) was then expressed in HEK-293 cells for the purpose of isolating highly purified RyR1 and interacting protein partners under non-denaturing conditions. Liquid chromatography mass spectrometry (LC-MS) was employed as a first screen in identifying these interacting proteins. Here, we identified calcium homeostasis endoplasmic reticulum protein (CHERP), as a protein that interacts with RyR1 and regulates its Ca 2ϩ release channel function.

EXPERIMENTAL PROCEDURES
Vector Construction-The cDNA encoding the cytosolic N-terminal fragment of RyR1, described previously (10), was inserted into the Qiagen pQE expression vector system containing the dual tandem affinity tags encoding 6ϫ-histidine and streptavidin-binding peptide to form the C-terminal His 6 tag. The His 6 -tagged cytosolic RYR1 cDNA was then excised with XbaI and HindIII and treated to form a blunt end and ligated in-frame into the pcDNA3.1 vector via its EcoRV site.
Expression of Cytosolic RyR1 in HEK-293 Cells and Preparation of the Protein Extract-The His 6 -tagged RyR1 protein was expressed in HEK-293 cells by transfection with 10 g of the cDNA construct per 100-mm plate using the standard calcium phosphate transfection protocol (11). Cells were harvested 48 h after transfection in an ice-cold solution of PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) containing 5 mM EDTA. The cells were then pelleted by centrifugation at 4100 rpm for 5 min at 4°C and resuspended and washed in ice-cold PBS alone. After removal of the supernatants, the cells were resuspended in 2 ml of lysis buffer (5% sucrose, 150 mM NaCl, 50 mM Tris-Hepes, pH 7.0, 1 mM PMSF, 1ϫ EDTA-free protease inhibitor mixture tablets (Roche Applied Science), 0.01% Triton X-100, and 5 mM ␤-mercaptoethanol. The cells were lysed using 25 strokes in a loose-fitting Dounce homogenizer. To remove cell debris, the lysate was then centrifuged at 8000 rpm for 20 min at 4°C. The supernatant was retained for analysis.
Purification of RyR1 Complexes Using Ni-NTA Resin-Two l of 50% Ni-NTA resin slurry per 1 plate of transfected HEK-293 cells was added to the protein extract, and the solution was rotated for 2 h at 4°C to allow the tagged proteins to bind the Ni 2ϩ via the His 6 tag. Proteins bound to the Ni-NTA resin were then separated from the flow-through by centrifugation (4100 rpm, 5 min, 4°C) and washed 3 times in 1 ml of ice-cold Ni-NTA binding buffer (lysis buffer with 25 mM imidazole added) by rotation (5 min; 4°C) followed by centrifugation (4100 rpm; 5 min; 4°C). Bound proteins were eluted twice for 1 h with 100 l of ice-cold elution buffer (binding/lysis buffer with 500 mM imidazole). Protein Sample Preparation and Proteomic Analysis-Aliquots containing 100 g of total protein from the elution fraction were prepared for trypsin digestion by the addition of ammonium bicarbonate, pH 8.5, to 50 mM and CaCl 2 to a final concentration of 1 mM in a total volume of 180 l. Samples were incubated overnight at 37°C with 0.02 mg/ml proteomics grade trypsin (Roche Applied Science) as described previously (12,13). Before application to the LC-MS columns, the digested peptides were solid-phase extracted using OMIX C-18 pipette tips (Varian Inc.) according to the manufacturer's protocol.
Comprehensive gel-free shotgun sequencing of tryptic digests was performed essentially as described previously (13,14). Briefly, for peptide separation, individual samples were first loaded onto microcapillary fused silica columns with an internal diameter of 75 m packed with 7 cm of reversed phase C18 resin (Magic C18). The columns were aligned with an LTQ linear ion trap mass spectrometer, and the peptides were eluted using a 2-h water/acetonitrile gradient and ionized via electrospray ionization. The tandem mass spectra generated were matched to peptide sequences in the human IPI protein sequence data base using the X!Tandem algorithm. Only proteins identified with at least two unique peptides were accepted for further experimental consideration.
Immunoblot Analysis-Immunoblotting was performed on protein fractions collected throughout the purification process. Thirty g of protein from the cellular lysate, flow-through, final wash, and elution fractions from RyR1 Ni-NTA purifications were subjected to standard Western blotting techniques (15). The blots were incubated first with the following commercial antibodies to target specific proteins: mouse monoclonal against RyR1 (Affinity Bioreagents), rabbit polyclonal against CHERP (Abcam), rabbit polyclonal against endoplasmic reticulum Golgi intermediate complex 53 (ERGIC-53) (Sigma), goat polyclonal against T-complex protein (TCP) (Abcam), mouse monoclonal against DHPR (Affinity Bioreagents), and rabbit polyclonal against phosphorylase kinase ␣-subunit (PHK-␣) and ␤-subunit (PHK-␤) (Abcam). Blots were then incubated with the corresponding horseradish peroxidase-conjugated secondary IgG. Finally, the blots were incubated with the SuperSignal ultra chemiluminescent substrate (Pierce) for 5 min and then exposed to a BioMax film (Eastman Kodak Co.).
Rat Skeletal Muscle Preparation and Confocal Immunofluorescence Microscopy-Skeletal muscle soleus fibers were isolated from the hind limbs of adult female Sprague-Dawley rats after euthanization via CO 2 asphyxiation. Immediately after isolation, the tissue was incubated in excess ice-cold PBS for 30 min before fixation in 2% paraformaldehyde for 30 min at 4°C. Two 1-ml washes were then performed with freshly prepared permeabilization buffer (0.2% Tween 20, 0.5% Triton X-100 in 1ϫ PBS) at 4°C for 15 min each. Immunofluorescence was performed on the skeletal muscle tissue as described by Sharma et al. (16).
Ca 2ϩ Transient Analysis-A microfluorimetry system was used to measure the effect of caffeine on Ca 2ϩ release in HEK-293 cells. Cells were transfected at ϳ50% confluence with fulllength RyR1 cDNA via the calcium phosphate method (11). The medium was changed after 24 h, and 2 h later siRNA was transfected using Lipofectamine reagent (Invitrogen). Ca 2ϩ photometry was performed on intact cells 48 h after siRNA transfection. The cells were loaded with 2 M Fura-2 AM (Invitrogen) for 1 h at pH 7.4 and room temperature in a physiological medium. The glass coverslips were placed in a holder on the stage of an inverted Olympus IX87 microscope. The samples were excited alternately at 340 and 380 nm with a dual monochromator. The emitted fluorescence was fed into a charge-coupled device camera. Acquired digital images were analyzed with the Quorum Analysis software.
For the 340/380-nm ratio acquisitions, cytosolic Ca 2ϩ concentrations were read over a period of 60 s with fluorescence measurements taken every 3 s, with 5-15 cells measured per run. At 30 s, the appropriate concentration of caffeine was added to induce Ca 2ϩ release. No attempt was made to wash out caffeine during the procedure.
Statistical Analysis-Data are reported as the mean Ϯ S.D. Statistical differences were evaluated by analysis of variance. A p value Ͻ0.05 was considered statistically significant.

Expression and Purification of Cytosolic RyR1
Proteins in HEK-293 Cells-To test for expression of the cytosolic RyR1 construct, confocal microscopy imaging was performed on cytosolic-RyR1-transfected HEK-293 cells (Fig. 1A) using the 34C antibody against RyR1. Phase contrast imaging of the same cells ( Fig. 1B) showed good transfection efficiency. WT HEK-293 control cells showed no signal (data not shown); thus, it can be deduced that the proteins visualized in Fig. 1 were generated from the cDNA construct and contain the His 6 affinity tag.
To ensure that an overexpressed protein of the size of cytosolic RyR1 could be purified efficiently, we examined eluants from Ni-NTA columns in Coomassie-stained gels (Fig. 1C). Purified cytosolic RyR1, indicated by a red arrow, was accompanied by multiple additional protein bands (black arrows), representative of co-purified potential RyR1 interacting proteins. To ensure that the protein detected by the red arrow in Fig. 1C was RyR1, immunoblot analysis was performed (Fig.  1D). The immunoblots show that a significant amount of the expressed recombinant protein was purified and concentrated in the elution fraction.
Identification of RyR1 Interacting Partners via LC-MS-A total of 9 Ni-NTA purifications were performed from HEK-293 cells, and the elution fractions were analyzed by MS. Of these, three were RyR1 purifications, three were negative control bead purifications, and three were His 6 -tagged DPYSL3 protein control purifications. This control was selected because DPYSL3 is a soluble cytosolic protein with few Gene Ontology terms associated with Ca 2ϩ signaling and EC coupling (17,18). In every experimental run, only proteins that were supported by at least 2 unique peptides were accepted for further consideration. A total of 703 proteins were identified from all 9 purifications including controls and RyR tags. These proteins are represented in a heat map (Fig. 2, left) prioritized by ascending spectral count in the RyR1 MS runs.
Of the 703 proteins detected, 323 were identified in at least 1 of the 3 RyR1 purifications, and the remaining 380 proteins were not considered further. To identify RyR1 interacting proteins with higher confidence, the subsets of identified proteins were filtered using two criteria. When proteins were found only in RyR purifications and not in controls, they were included only if they were present in at least two of the three RyR1 runs but not in any of the six control runs. For proteins that were found in both control and RyR purifications, spectral counts were averaged for each identified protein over the three RyR1 runs and for the six control runs to generate an enrichment value over controls. Only those proteins that were enriched by a factor of four were included. Forty-nine proteins passed these criteria, and they are represented in Fig. 2 (right) and listed in Table 1.
To further filter this list, each protein was subjected to a literature search to assess its potential as an RyR1 interacting protein. Proteins classified as originating in the ribosome, the mitochondria, the spliceosome, or the histone fraction or as being associated with metabolism or heat shock were eliminated. This resulted in only five candidates passing these criteria. They were CHERP, TCP-␣, PHK, ERGIC-53, and MYCBP2. MYCBP2 is an ubiquitin ligase (19,20). It was removed from consideration, as ubiquitin-related pathways are not likely to be involved in cellular Ca 2ϩ homeostasis. CHERP, TCP-␣, PHKB, and ERGIC-53 were selected for subsequent interaction validation studies.
CHERP, is an integral sarco/endoplasmic reticulum (SR/ER) membrane protein. It is a 916 (human)-and 936 (mouse)amino acid protein. The human sequence is predicted to contain 1 luminal to cytosolic, transmembrane (TM) sequence near its N terminus (amino acids 233-252; predicted with "very high certainty" on the basis of a high hydrophobicity score of 1.25 using the Kyte-Doolittle scale (21)). It also has a second potential TM sequence (amino acids 641-659; predicted with "putative" probability based on a 0.78 hydrophobicity score) (22). This domain structure would suggest a luminal N terminus of 230 amino acids and either a long cytosolic domain of 664 amino acids (if 1 TM sequence) or 388 amino acids (if 2 TM sequences). A cytosolic coiled-coil domain at amino acids 290 -312 is also predicted.
Because CHERP has been described as playing a role in cytosolic Ca 2ϩ cycling in various cell types (21,23), it was selected for further study. TCP-␣ is a molecular chaperone and a member of the chaperonin-containing TCP1 complex (24). Because it is unknown how a protein channel the size of RyR1 is organized and folded in vivo, TCP-␣ was deemed a potential candidate. Phosphorylase kinase is a serine/threonine-specific protein kinase composed of several subunits, including calmodulin (the ␦ subunit) (25). The phosphorylation of various RyR1 residues, in particular Ser-2843, activates RyR1 (26). Thus, the rationale for including PHK as a potential RyR1 interacting protein was its potential for phosphorylating RyR1. ERGIC-53 is a type 1 transmembrane glycoprotein receptor protein localized

RyR1 Interacts with CHERP
to the ER-Golgi intermediate compartment that is involved in protein trafficking from the ER to the Golgi. Its affinity for substrates is dependent on cytosolic Ca 2ϩ levels (27).
Immunoblot Analysis of Ni-NTA Eluates to Verify RyR1 Pulldown of Candidate Proteins-Western blotting was performed on 25 g of protein from the flow-through, first wash, final wash, and eluant fractions of the Ni-NTA purification step. Using antibodies against the four candidate proteins, we verified the presence of RyR1 interacting candidates in the purified samples together with RyR1 (Fig. 3A). ERGIC-53, CHERP, TCP, and PHK-␣ were all detected by immunoblotting after purification of affinity-tagged RyR1 on Ni-NTA resin. PHK-␤ staining was found to be unreliable and nonspecific and not pursued (results not shown). However, when the same experiments were performed in untransfected HEK-293 cells, faint bands for ERGIC-53, TCP, and PHK-␣ elutions indicated that those proteins were bound to the Ni-NTA resin in the absence of RyR1. Accordingly, they were eliminated as potential RyR1 interacting candidates. CHERP, however, was not found in any control blots.
We were not able to perform successful co-immunoprecipitation assays from mouse or rabbit skeletal muscle either in a forward or reverse direction with any of the available RyR antibodies under numerous experimental conditions. These results are consistent with the extremely limited data concerning RyR immunoprecipitations in the literature.
To ensure the effective suppression of CHERP levels, Western blotting was used to compare protein samples from HEK-293 cells expressing either RyR1 alone, RyR1 together with CHERP siRNA, or RyR1 together with a scrambled CHERP siRNA. Fig. 3B shows that RyR1 was expressed at comparable levels in all three treated cells and that endogenous CHERP was expressed at comparable levels in the cells expressing RyR1 or RyR1 plus scrambled siRNA. However, in those cells treated with CHERP siRNA, the expression of endogenous CHERP was reduced dramatically.
To ensure that CHERP is expressed in WT skeletal muscle, we analyzed WT mouse skeletal muscle using Western blotting against protein samples from two different mice. Fig. 3C FIGURE 2. Proteins identified by mass spectrometry from RyR1 purifications. Left, a heat map representation of 703 proteins identified by mass spectrometry in all tandem affinity-purified samples from HEK-293 cells is shown. Color intensities depict total spectral counts (as a function of log 10). Right, shown is a specific cluster from the left heat map, identifying filtered proteins selected in one of two ways; 1) proteins identified in more than one RyR1 experiment and absent from all controls, or 2) Proteins whose average spectral count over all RyR1 experiments exceeded the average spectral count over controls by at least a factor of 4. This resulted in the selection of 49 proteins. MAY 13, 2011 • VOLUME 286 • NUMBER 19

RyR1 Interacts with CHERP
shows that CHERP is present in mouse skeletal muscle in the same samples that express RyR1 and skeletal myosin heavy chain.
Immunofluorescence Co-localization of CHERP and RyR1 in Rat Skeletal Muscle-In skeletal muscle, RyR1 is retained in the junction terminal cisternae of the SR membrane (10). Through immunofluorescence staining, RyR1 can be resolved as double rows of punctate immunosignals, with each spot representing a single triad structure. The Z-line lies between the RyR1 doublet (28). In skeletal muscle co-stained with CHERP and RyR1 antibodies, co-localization and partial overlap pattern was evident (Fig. 4A). Co-localization and partial overlap was observed at both of the RyR1 double rows and was most consistent on the side facing the Z-line throughout the longitudinal section. This gave the appearance of two RyR1 rows, split by the Z-line, each with a CHERP row proximal to the Z-line. Fig. 4B shows the three-dimensional reconstitution of a Z-stack of two-dimensional images from mouse skeletal muscle tissue co-stained with RyR1 (green) and CHERP (red) antibodies. The Imaris analysis software allowed us to calculate the percentage of co-localization and overlap observed in a three-dimensional image. The co-localization software employed a computational algorithm to determine the presence of each signal in one voxel (a three-dimensional pixel), thus determining the percentage of co-localized voxels containing both green and red signals. Through this analysis, 22% of the voxels within the region of interest were found to colocalize. To ensure that colocalization results were not affected by the nonspecific binding of fluorescent secondary antibodies, we incubated rat soleus muscle with Alexa 488 and 633 secondary antibodies followed by confocal imaging. Under these conditions, co-staining could not be resolved, so we could not reconstruct three-dimensional images from a Z-series of images (data not shown).
Our three-dimensional pattern of co-localization of RyR1 and CHERP (Fig. 4B) indicates that CHERP and RyR1 sit adja- cent to each other in the ER/SR membrane. CHERP is a predicted to be an integral ER membrane protein with at least 1 TM sequence and a large cytosolic domain (21). With this in mind we generated a schematic model of interaction between these two proteins (Fig. 4C).
To further validate our three-dimensional fluorescence data, we generated a three-dimensional reconstruction of mouse skeletal muscle tissue RyR1 (green) co-stained with DHPR (red), a protein known to interact with RyR1 (Fig. 4D). The co-localization algorithm determined that 28% of the voxels were colocalized and that the staining pattern was stacked. This is exactly what is predicted, as the DHPR is located in the transverse tubular membrane, whereas RyR1 is embedded in the SR membrane, as indicated in Fig. 4C.
Ca 2ϩ Transient Analysis of HEK-293 Cells Expressing Fulllength RyR1 Alone and with siRNA Suppression of CHERP-To study the effect of CHERP suppression on Ca 2ϩ transients in HEK-293 cells expressing full-length RyR1, we acquired 340/ 380-nm fluorescence ratios from cells treated with caffeine, an RyR1 activator (Fig. 5A).
The amplitudes of the Ca 2ϩ transients seen in Fig. 5B represent the increase in the fluorescence ratio over base-line values upon stimulation with 5 mM caffeine. The fluorescence ratio is directly proportional to the level of intracellular Ca 2ϩ . Each condition was repeated 3 or more times with emissions from clusters of 5-12 cells measured in each repeat. These amplitudes are quantified in Fig. 5C. Fluorescence amplitude was calculated by dividing each point throughout the curve by the FIGURE 3. Immunoblot analysis of His 6 -tagged RyR1 overexpression, CHERP siRNA knockdown, and endogenous expression of RyR1 and CHERP in mouse skeletal muscle. A, fractions analyzed were the cell lysate, the flow-through, or unbound fraction, which is the lysate after incubation with Ni-NTA resin, the final wash, and the fraction eluted with 500 mM imidazole. DHPR, known to interact with RyR1, was used as positive control. B, HEK-293 cells were transfected with full-length RYR1 cDNA 24 h after plating and 2 h later with either blank Lipofectamine (ϩRyR1) or siRNAs, knocking down either CHERP (ϩRyR1 ϩ CHERP) or scramble siRNA (ϩRyR1 ϩ SCRAM). Twenty g of protein from each sample were run on SDS-PAGE and analyzed by Western blotting. C, WT mouse skeletal muscle was harvested, homogenized, and centrifuged at 8000 rpm to obtain a soluble cell lysate. Twenty g of protein were run on SDS-PAGE and analyzed by Western blotting to determine endogenous levels of RyR1 and CHERP in WT skeletal muscle. B, three-dimensional reconstruction of a two-dimensional Z-stack of images illustrates the subcellular distribution of endogenous CHERP and RyR1 in rat soleus skeletal muscle (left panel). Analysis of images for three-dimensional colocalization shows that 22% of the voxels within the region of interest seen above were colocalized. Right panel, colocalized voxels only. C, shown is a schematic representation of co-localization between RyR1 and CHERP. D, shown is a three-dimensional reconstruction of a two-dimensional Z-stack of images illustrating the subcellular distribution of endogenous RyR1 and DHPR in mouse soleus skeletal muscle (left panel). Analysis of images for three-dimensional colocalization showed that 28% of the voxels within the region of interest seen above were colocalized. Right panel, colocalized voxels only. minimum fluorescence, thus generating a base-line value of ϳ1.0. The amplitude thus represents the percentage change in the fluorescence ratio.
The percentage increase in the fluorescence ratio in HEK-293 expressing full-length RyR1 in the presence of endogenous CHERP (36.1 Ϯ 4.8%; p value Ͻ0.05) was significantly higher than the percentage increase in fluorescence observed when endogenous CHERP expression was suppressed by siRNA (8.1 Ϯ 1.0%). In all runs, after the caffeine-induced Ca 2ϩ release event, a decrease was observed in the 340/380-nm ratio, although the base line remained elevated, as we did not attempt to wash out added caffeine during this period. This represents subsequent Ca 2ϩ reuptake by the ER after the Ca 2ϩ release event and establishes a Ca 2ϩ transient. In every run the uptake or decrease in 340/380-nm ratio was in the range of 25-40% of the initial increase in 340/380-nm ratio. This response to caffeine-induced Ca 2ϩ release is consistent with reports in the literature.
When we measured caffeine dose dependence curves for HEK-293 cells expressing RyR1 and RyR1 with suppressed CHERP, we observed a decrease in caffeine maximal efficacy on Ca 2ϩ release from RyR1 in those cells with low expression of CHERP (Fig. 5C). Cells were treated across a range of caffeine concentrations up to 12 mM, the upper limit of an RyR1 response to caffeine-induced Ca 2ϩ release (29). At 12 mM there was a significant difference in the percentage of maximal fluorescence ratio observed in cells with low expression of CHERP (p Ͻ 0.05, analysis of variance).

DISCUSSION
In this study we screened for RyR1 interacting proteins that might affect RyR1 function by overexpressing and purifying the soluble, cytosolic domain of RyR1 in HEK-293 and determining by mass spectrometry, immunoblotting, and immunofluorescence microscopy whether interacting proteins might co-purify and colocalize with RyR1. Our studies show that CHERP co-purifies with RyR1 and that it co-localizes with RyR1 in endogenous rat skeletal muscle. Subsequently, we showed that a decrease in the expression of endogenous CHERP in HEK-293 cells expressing full-length, functionally active RyR1 reduced Ca 2ϩ efflux from the ER via RyR1, suggesting that protein-protein interactions between CHERP and RyR1 contribute to the activity of RyR1 Ca 2ϩ release channels and, by analogy, may be involved in the regulation of EC coupling in muscle cells.

Expression and Purification of Cytosolic RyR1 Proteins and Co-purification of Interacting Proteins from HEK-293 Cells-
To identify novel RyR1 protein interactions, metal affinity chromatography was applied to extracts of HEK-293 cells expressing a soluble, truncated form of RyR1. This method was chosen for its efficiency, potential for high-throughput application, and capacity for purifying protein complexes from in vivo systems. HEK-293 cells provided a large cell mass, an easily transfected cell line, and generation of a high overall yield of soluble bait and interacting proteins. Concerning the RyR1 purification itself, the recovery and concentration of bait protein from the cell lysate was performed successfully; in the RyR1 purifications and subsequent MS runs, RyR1 spectral counts greatly exceeded those of any other protein. show that lowered CHERP levels induce a marked decrease in the amplitude of the Ca 2ϩ transient when the cells were stimulated with caffeine. B, the percentage increase of the 340/380 nm ratio over base-line values after the addition of 5 mM caffeine was calculated; after siRNA knockdown of CHERP, Ca 2ϩ release was significantly reduced (*, p Յ 0.05). C, dose-response curves were obtained by Ca 2ϩ photometry of HEK-293 cells transfected with wild type RYR1 cDNA or with wild-type RYR1 cDNA followed by transfection with CHERP or scrambled siRNA. The amplitude of each peak caffeine response was normalized to the amplitude of the peak caffeine response. Changes in fluorescence ratio are presented as ⌬r ϭ (R Ϫ R min )/(R max Ϫ R min ), where R refers to the 340/380 nm fluorescence ratio at each caffeine concentration, and R min and R max refer to the fluorescence ratio under resting conditions and at the highest response to caffeine. R is plotted as a function of caffeine concentration. Upon stimulation with 12 mM caffeine, the upper limit of RyR1 caffeine response, there was a significant reduction in fluorescence response in cells suppressing CHERP (*, p Յ 0.05).
Our stringent data-filtering criteria removed some known RyR1 interactors from our final candidate list. For example, DHPR, which physically interacts with RyR1 in skeletal muscle (30), was excluded because it appeared in only one RyR1 mass spectrometry run. FKBP1A, another known interacting protein (7), was retained because it was identified in 2 runs, and FKBP1A spectral counts were, on average, ϳ4-fold enriched over controls.
A subset of 49 potential RyR1 interacting proteins was identified after the filtering of the MS data. Individual analysis of these proteins revealed that many were involved in biological processes that are not directly related to RyR1 and Ca 2ϩ regulation, so they were eliminated as potential interacting proteins. The identification of these proteins on the basis of their known functions probably represents nonspecific binding of contaminant proteins to bait-binding resin, which is a common limitation of affinity chromatography (31). Washing the Ni-NTA resin throughout the purification process did not remove all contaminants, allowing these proteins to pass even stringent criteria for MS identification. Many contaminant proteins are commonly found in proteomics experiments. They are present either by accident or through unavoidable contamination of protein samples. These proteins are listed online in the contaminant repository of adventitious proteins (cRAP; The Global Proteome Machine Organization).
CHERP as a Potential RyR1 Interacting Protein-Of the 49 RyR1 co-purified proteins that are candidates for interaction with RyR1, a select number were identified in the literature as being involved in Ca 2ϩ homeostasis and muscle processes, allowing us to filter the MS data list down to a handful of proteins for further analysis. These proteins, PHK, TCP-␣, ERGIC-53, and CHERP, were subjected to immunoblot analysis to determine whether they could be identified in the eluant fractions of RyR1 purifications. Although all of the proteins were identified in these fractions, only CHERP was not found in control experiments where Ni-NTA resin was incubated in WT HEK-293 cellular lysate. This suggests that these candidate proteins bound to the Ni-NTA resin non-specifically and were contaminant proteins.
CHERP was first identified through its interaction with a monoclonal antibody raised against inositol 1,4,5-trisphosphate receptor-rich dense-tubular-system membranes of platelets (23). This antibody inhibited Ca 2ϩ release by inositol 1,4,5trisphosphate receptor from internal membrane vesicles derived from platelets, cerebellum, smooth muscle, sea urchin eggs, and human erythroleukemia cells (23). CHERP is found in many cell types, including HEK-293 cells (this study) and mammalian skeletal muscle. It has been found to play a role in the regulation of intracellular Ca 2ϩ mobilization. Our study has identified CHERP as a RyR1 interacting and regulatory protein, which may be involved in the regulatory mechanism of EC coupling.
More in-depth, functional work has been performed with CHERP in Jurkat cells. Transfection of Jurkat cells with a lacregulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca 2ϩ levels caused by phytohemagglutinin and thrombin (21). A 50% fall of CHERP decreased the phytohemagglutinin-induced rise of the cytoplasmic free Ca 2ϩ concentration, but Ca 2ϩ influx was unaffected. Furthermore, cell proliferation was slowed. Although these findings provide further evidence that CHERP is an important component of ER Ca 2ϩ mobilization and that its loss impairs Ca 2ϩ -dependent biochemical pathways and progression through the cell cycle, no mechanism of these effects was ever offered. However it was shown that Ca 2ϩ influx from extracellular spaces was not affected.
Jurkat cells express RyR1 according to an earlier study elucidating the expression of RyR isoforms in immune cells (32). It was found that 4-chloro-m-cresol, a specific activator of RyR1, induced Ca 2ϩ release, thereby confirming functional expression of RyR in Jurkat cell lines. Because Ca 2ϩ influx was unaffected and because Jurkat cells express RyR1, it is plausible that CHERP suppression was responsible for lower cytosolic Ca 2ϩ levels because of reduced RyR1 activity.
In the present study CHERP suppression affected cytosolic Ca 2ϩ levels in a manner similar to that observed in the O'Rourke et al. study (21). Here, siRNA knockdown of CHERP resulted in a reduction of total Ca 2ϩ release from the ER by 56%. Based on the 4-fold enrichment of CHERP spectra in MS-analyzed RyR1 purifications, in our CHERP-RyR1 co-localization results, which show subcellular colocalization of CHERP and RyR1 in skeletal muscle SR (22% co-localized voxels, when analyzed by three-dimensional software), we suggest that CHERP interacts directly with RyR1 to promote Ca 2ϩ release in skeletal muscle cells.
Cytosolic Ca 2ϩ levels are determined by numerous factors, including Ca 2ϩ uptake by sarco/endoplasmic reticulum Ca 2ϩ -ATPase and extracellular Ca 2ϩ release by the plasma membrane Ca 2ϩ ATPase and the sodium calcium exchanger and the NCX as well as Ca 2ϩ uptake and release by the mitochondria. The properties of these channels, however, leave RyR as the only component of Ca 2ϩ flux capable of generating the magnitude of Ca 2ϩ release observed in our Ca 2ϩ transient results. Thus, regulation of RyR1 function by CHERP of the magnitude that we observed would not be trivial in the context of Ca 2ϩ handling in muscle cells.
Potential Physiological Relevance of a CHERP-RyR1 Interaction on EC-coupling Regulation-The amino acid sequence of CHERP described by O'Rourke et al. (21) suggested an integral ER membrane protein that appears to contain several relevant domains that might interact with RyR1. Recent analyses of the topology of CHERP suggest that it contains either 1 or 2 TM regions (233-252 and 641-661) (22). As a result, 1 or 2 TM sequences would lead to either a 388-or 664-amino acid cytosolic domain. Because we used a large cytosolic domain of RyR for interaction analysis, we predict that CHERP-RyR1 interactions occur in the cytosol via cytosolic domains. It is also likely that CHERP interacts with RyR1 at or near the RyR1 membrane sector and not at the most distal cytosolic portion, believed to interact with the DHPR.
In future studies, it will be important to assess the function of CHERP through muscle cell lines for both the RyR1 (skeletal) and RyR2 (cardiac) isoforms. It will also be important to localize and characterize CHERP-RyR1 binding sites and to define the regulatory mechanism whereby CHERP exerts its effects on RyR1 Ca 2ϩ release channel function. At this stage we cannot conclude whether CHERP knockdown results in direct inhibition of Ca 2ϩ release channel function or, alternatively, whether our results reflect depletion of the ER/SR Ca 2ϩ store induced by loss of CHERP. Given the relative abundance of CHERP in skeletal muscle, as indicated in our morphological studies, and its intimate proximity to RyR1, it is probable that CHERP plays a significant role in ER/SR function.