Activation of Ca transport in cardiac microsomes enriches functional sets of ER and SR proteins

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Cala, Nicholas J. Carruthers, Paul M. Stemmer, Zhenhui Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2557992/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Apr, 2023 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract The importance of sarcoplasmic reticulum (SR) Ca-handling in heart has led to detailed understanding of Ca-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Analyses of protein enrichments from the 3 membrane preparations (crude microsomes, MedSR, and HighSR), showed that only a third of microsomal proteins in heart, or 354 proteins, were enriched ≥2.0-fold in SR. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca leak (ryanodine receptor) co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins from other putative ER/SR subdomains also showed characteristic distributions among SR subpopulations. We conclude that active Ca loading of cardiac microsomes, reflecting the combined activities of Ca uptake by SERCA, and Ca leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR. cardiac sarcoplasmic reticulum endoplasmic SERCA ryanodine receptor proteome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The basic morphology of the endoplasmic reticulum (ER) in muscle has been known since the initial electron microscopic studies in the 1950’s by Bennett and Porter [ 1 ] and Palade and Porter [ 2 ], when it was termed the sarcoplasmic reticulum (SR) 1 , given its unique structure that repeats across all sarcomeres in the myocyte. Studies over many decades have focused on its Ca handling properties, beginning with extensive characterization of the important Ca translocating ATPase activity, now termed SERCA, for SR, ER-Ca-ATPase [ 3 , 4 ]. Much later, a ryanodine-sensitive Ca-releasing activity was identified [ 5 – 7 ]. Extensive analyses of Ca transport in membrane preparations from skeletal muscle also led to established membrane subfractionation protocols, and isolation of membranes of distinct densities that contained different protein composition. Meissner [ 8 ] reported the first fractionation of skeletal muscle membranes by isolating a fraction of heavy vesicles that contained the electron-opaque protein polymer calsequestrin [ 9 ], and later shown to contain foot processes consistent with morphological depictions of junctional SR [ 10 ]. The lighter membranes showed less of the dense luminal protein content, and was concluded to contain free (non-junctional) SR vesicles [ 8 , 9 ]. A technique for subfractionation of microsomes from heart tissue was later developed that took advantage of the ability of cardiac SR microsomes to concentrate Ca oxalate within their lumens [ 11 – 13 ]. While these studies were originally developed to biochemically separate sarcolemma and SR, subsequent work [ 5 ] showed that the Ca oxalate loading had actually produced two distinct types of SR membranes, with roughly half of membrane protein in the densest membrane fraction. And though it was known by then that the drug ryanodine could stimulate Ca accumulation into SR membrane vesicles at high concentrations, these dense membranes were ryanodine insensitive [ 5 ]. In contrast, a second membrane fraction of lower density (unable to traverse a 1.5 M sucrose cushion) was highly sensitive to ryanodine [ 5 ]. Indeed, by inhibiting Ca leak, 0.3 mM ryanodine added during membrane isolation converted SR membranes of lower density into high density (high Ca oxalate) vesicles [ 14 ]. The densities of SR membrane vesicles, as well as the distribution of SR proteins between the two subfractions, are regulated by the relative levels of SERCA and ryanodine receptor (RyR) contained in the fragmented SR patches. Several studies have used SERCA-positive membrane subpopulations to either validate the identity of a putative junctional SR protein [ 5 , 15 – 17 ], or non-junctional SR protein [ 14 , 18 – 21 ]. In this study, we determined the complete protein compositions of membrane vesicles generated by SERCA-depending Ca oxalate loading. We found 354 proteins that were enriched as a result of SERCA activation. In addition, a co-enrichment of functionally related proteins resulted from their similar distribution among membrane patches, and reflect the enrichment of ER/SR subdomains in close proximity to SERCA2 and RyR2 activities in intact cardiomyocytes. Methods Preparation of Crude Cardiac Sarcoplasmic Reticulum Vesicles - Cardiac microsomes were isolated from female mongrel dog left ventricular tissue, and loaded with Ca oxalate as previously described, with minor modifications [ 5 ]. Briefly, heart tissue was homogenized in 10 mM NaCO 3 at 1:20 dilution (buffer volume/tissue wet weight). Cardiac microsomes were isolated by differential centrifugation, isolating microsomes between 10,000–75,000 x g max . In contrast to the previously published method, a final wash of the pellet in 0.6 M KC1, 30 mM histidine, pH 7.0, was not carried out. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 − 23, revised 1996). Animal research was approved by the Wayne State University Animal Investigation Committee (protocol #A 04-02-13). Ca oxalate loading of SR - Loading of crude cardiac microsomes, 75 mg of protein were resuspended in 40 ml of an ice-cold medium containing 50 mM histidine, 100 mM KCI, 65 mM MgCl 2 , 60 mM Na 2 ATP, 25 mM Tris/EGTA, 20 mM CaCl 2 , and 5 mM Tris oxalate (pH 7.1). The suspension was rapidly warmed to 37 o C to initiate active and rapid Ca uptake, and the incubation was conducted for 10 min with 5 mM additional Tris/oxalate added after the first 5 min of incubation. The suspension was then immediately centrifuged at 4 ºC for 30 min at 100,000 x g max . The resulting brownish membrane pellet included a central white region, indicative of the Ca oxalate precipitate inside the highest density membranes. The whitish center was not present if ATP was not included in the Ca oxalate loading step (Fig. 1 A). Isolation of medium and high density membrane vesicles after Ca oxalate loading – Subfractionation of Ca loaded membranes was carried out essentially as previously described [ 5 ]. Briefly, membrane pellets were resuspended in 0.25 M sucrose containing 300 mM KC1, 50 mM sodium pyrophosphate, and 100 mM Tris (pH 7.2). This material was layered over a discontinuous sucrose gradient of 0.6 M, 0.8 M, 1.0 M, and 1.5 M sucrose dissolved in the same buffer. After centrifugation at 125,000 x g max for 2 h, membranes were collected from both the 1.5 M sucrose cushion (MedSR) or as the white pellet at the bottom of the gradient (HighSR). These two subfractions were not present in membranes not Ca loaded (Fig. 1 B). MedSR was diluted with 4 volumes of ice-cold H 2 O, and then sedimented at 105,000 x g max for 60 min. MedSR and HighSR were resuspended in 0.25 M sucrose, 10 mM histidine, and stored frozen at -20 o C. Protein was determined by the method of Lowry et al. (17). Preparation of protein samples for mass spectrometric analysis - To evaluate the protein composition of the two SERCA-positive membrane fractions, relative to the crude cardiac membranes, we analyzed exactly 20 µg each of MVs, MedSR, and HighSR membranes by SDS-PAGE. The wet gel was stained with SPYRO Ruby red to visualize the protein separations qualitatively (Fig. 1 C). Tryptic digestion of SR samples - For in-gel digestion (GeLC-MSMS), each lane (crude cardiac microsomes (MV), MedSR, or HighSR membranes) was cut into 20 gel slices for improved sampling (Fig. 1 C). Gel slices were equilibrated in H2O, dried, and digested with trypsin added in dilute solution (1:10 w/w) and digested overnight at 37 o C. LC-MS/MS analysis and database search - Tryptic peptides were separated by reverse phase chromatography (Magic C18 column, Michrom), followed by ionization with the ADVANCE ion source (Michrom), and then analyzed in an LTQ-XL mass spectrometer (Thermo Fisher Scientific). For each MS scan, up to seven MS/MS scans were obtained using collision-induced dissociation. Data analysis was performed using Proteome Discoverer 1.1 (Thermo) which incorporated the Mascot algorithm (Matrix Science). The NCBI database was used against mammalian protein sequences and a reverse decoy protein database was run simultaneously for false discovery rate (FDR) determination. Secondary analysis was performed using Scaffold (Proteome Software). A fixed modification of + 57 on cysteine (carbamidomethylation) and variable modifications of + 16 on methionine (oxidation) and + 42 on protein N-terminus (acetylation) were included in the database search. Minimum protein identification probability was set at ≥ 95%, with 1 unique peptide at ≥ 95% minimum peptide identification probability. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, along with their relative levels within each of the three different membrane fractions, when levels were sufficient. Raw data and search results have been submitted to the ProteomeXchange via PRIDE with accession PXD022455 (Username: [email protected] , Password: G1wlk66N). Results And Discussion 1. Protein composition of crude cardiac microsomes An abundance index (A SR ) was calculated for each protein, calculated as the number of its peptide spectra, divided by protein molecular weight as a rough correction for available target peptides, then normalized to the spectral level for SERCA2, which we defined as A SR =100.0. A SR values are a crude surrogate for protein abundance, since factors unrelated to protein abundance will affect the quantifcation of tryptic peptides. Nonetheless, A SR values across sets of functionally related proteins can provide useful comparisons. In a preparation of cardiac microsomes (MVs), we found 1102 proteins (see Online Resource 1). The most abundant peptides from crude cardiac MV membranes were from known mitochondrial proteins. The three proteins with the highest A SR values in cardiac microsomes were ATP/ADP translocase and ATP synthase, α and β subunits, with known mitochondrial proteins accounted for 7 of the top 10 proteins. The other three major proteins among the top ten highest A SR values were SERCA2, a keratin 1 isoform, and cardiac α1 actin. The focus of our experiments, however, was not directed at crude cardiac microsomal proteins, but instead, at proteins that are enriched in cardiac SR. Of the top 10 microsomal proteins detected, only SERCA2 was enriched in the denser membrane vesicles that result when Ca transport is activated by addition of ATP. 2. Enrichment of membrane vesicles by SERCA activation The two enriched SR subfractions analyzed in this study have previously been described as junctional SR and free SR vesicles; however, with the much greater number of proteins identified through the use of our GeLC-MS/MS analysis, we have substituted the terms “medium-density SR membranes” (MedSR) and “high-density SR membranes” (HighSR), for these two membrane subpopulations (Table 1 ). This terminology allows us to discuss the larger collection of proteins, whether or not they participate directly in Ca handling functions. Comparing the abundance of each protein (A SR ) in MedSR and HighSR membranes with its abundance in MVs, we determined protein enrichments in SR (E SR ) (Table 1 ). Calculated from ratios of A SR values, the enrichment values were relatively unaffected by the sampling issues for any given protein. We used the value of E SR to define SERCA-positive SR proteins as those proteins that were enriched by SERCA activation at least 2.0-fold (E SR ≥ 2.0). For 354 SERCA-positive SR proteins, the enrichments varied, but the average enrichment was 8.0-fold (avg E SR = 8.0). Table 1 Key definitions and abbreviations used in this study Membrane preparations MV Crude cardiac microsomal v esicles MedSR Medium density Ca oxalate loaded SR, enriched in every junctional SR marker HighSR High(est) density Ca oxalate loaded SR, enriched in free SR (non-junctional) vesicles Parameter determined for each protein Range of values E SR Enrichment in SR (MedSR + HighSR)/ MV ≥ 2.0 for SR (average: 8.0-fold) E sub Enrichment in HighSR compared to MedSR ( HighSR – MedSR)/ (HighSR + MedSR) = 0.90 (90% higher in HighSR) = −0.90 (90% higher in MedSR) = 0.00 (equally distributed) A SR Spectral Abundance in SR (MedSR + HighSR) , divided by molecular weight, normalized to SERCA2 (= 100.0) 0.05–100.0 A SR (SERCA2) ≡ 100.0 In contrast to enrichment of SR proteins in total SR (HighSR plus MedSR), enrichments in HighSR versus MedSR membranes (E sub ), reflect differences in the way small membrane patches are pulled apart, yielding small vesicles with varying levels of Ca release activity (RyR) on top of their very active Ca accumulation. Thus, individual proteins that segregate with MedSR were more likely to contain membrane patches that contain RyR; that is, closer to junctional SR sites in vivo . To evaluate this distribution as a single number, we defined E sub as the difference between levels in the two SR fractions divided by the total (Table 1 ). Proteins more enriched in High SR yielded a positive number with a theoretical maximal value of 1.0, and proteins enriched in MedSR yielded a negative number with a theoretical maximal value of -1.0. Values actually ranged from + 0.90 to -0.90. 3. Characterization of SR proteins based upon enrichments among membrane subpopulations Of the top 10 microsomal proteins detected in our ventricular muscle sample, only one (SERCA2) was enriched by SERCA activation (E SR =3.4). Among 200 microsomal proteins with the highest A SR values (Fig. 2 A, blue bars ), only 40 proteins were enriched at least 2.0-fold by SERCA activation ( red bars ). All SR proteins previously identified as resident to cardiac SR using immunological methods were among the proteins enriched by SERCA activation in our study. For 14 of the best characterized SR proteins (Fig. 2 B, green bars ), the average fold enrichment in SR over crude cardiac microsomes (E SR ) was 5.0 ±1.6 (mean ±S.D.). When sorted based on their relative distribution between SR subcompartments (E sub ), all of the known junctional SR proteins were more enriched in MedSR (Fig. 3 A), consistent with the idea that MedSR membrane patches probably contain more RyR molecules, possibly combined with less SERCA. Luminal ER/SR proteins, on the other hand, were highly enriched in HighSR (except for calsequestrin-2). SERCA2, SERCA1, and phospholamban were more equally distributed among the membrane vesicles, consistent with a more equal activation of Ca loading in both SR populations. Despite the similar enrichments of known SR proteins, their relative abundances (A SR , Fig. 3 B) varied greatly, consistent with data widely known from protein staining of SDS-gels [ 15 , 18 ]. For the entire set of 354 proteins in SERCA-positive membranes, E sub spanned a continuous range of values from + 0.90 to -0.90, corresponding to proteins at a 90% greater level in HighSR, or in MedSR, respectively (Fig. 4 ). Interestingly, for sets of proteins with related function, E sub values were clustered together. 2, in support of the idea that membrane fragments are generated from ER/SR membrane patches that contain characteristic ratios of SERCA and RyR levels. 4. High abundance proteins illustrate wider functions in cardiac SR than only Ca handling A SR values are based upon the number of peptide spectra attributed to a particular protein, but are only semi-quantitative, as they assume similar coverage of every protein sequence by LC-MS/MS, which does not occur [ 22 ]. Yet, it was very interesting to look at the proteins with the highest A SR values, as it illustrates the scope of protein functions in cardiac SR. The 5 most abundant proteins in our SERCA-positive SR sample were SERCA2, desmin, sarcalumenin, phospholamban, and calsequestrin-2 (CSQ2), accounting for about a quarter of SR protein mass (percent of total spectra) (Fig. 5 ). SERCA2a and phospholamban are well known major constituents of cardiac SR [ 5 , 13 ], and, as expected, their very high A SR values were a predictable consequence of their activity in Ca oxalate loading of SR vesicles. Sarcalumenin is a known luminal constituent of cardiac SR membranes [ 18 , 23 ], but of uncertain function. It is comprised of two well known splice variants: one generally defined by its roughly 150-kDa apparent molecular weight on standard SDS-gels, and one defined by a roughly 53-kDa [ 24 ]. Desmin, a muscle-specific intermediate filament protein of striated muscle [ 25 ], was found at A SR levels comparable to SERCA2 (Fig. 5 ). Surprisingly high A SR values were also found for many proteins barely discussed in the cardiac research literature. For example, NADH-cytochrome b5 reductase gave the highest A SR value of a non-traditional cardiac SR protein ( A SR = 30.0; 6th highest) . Salviati et al. [ 26 ] in 1981 reported very high levels of this redox enzyme, as well as cytochrome b5 itself ( A SR = 5.1; 69th highest) , in the SR of slow skeletal muscle. A prominent escort protein for rab proteins, PRA1 (prenylated rab acceptor 1), was prominently detected ( A SR = 25.0, 7th highest ). The presence of abundant histone H4 ( A SR = 21.0, 8th highest ), and other less abundant histones (Table 2, Appendix ) may suggest a role as a secreted antimicrobial protein [ 27 , 28 ] or copper reductase enzyme [ 29 ]. The high level of CDP-diacylglycerol-inositol 3-phosphatidyltransferase, isoform 1 (CDITP, A SR = 20.7, 9th highest) , was consistent with early studies by Kasinathan and Kirchberger [ 30 ]. Interestingly, the SR protein with the next highest A SR (VAP-B) is a prominent intra-organellar tethering protein that also plays a putative role in the transfer of synthesized PI to the plasma membrane. Both VAP-B and VAP-A (A SR = 18.5 and 11,3, 10th and 24th highest, resp.) were among multiple ER tethering proteins present [ 31 – 39 ] (Table 3 ). The combined actions of these abundant ER/SR proteins may promote new plasma membrane sites for PI signaling activity in heart, as well as sites for junctophilin-2 binding and junctional SR formation [ 40 – 43 ]. Extended synaptotagmins, detected at low A SR levels, can also transport glycerolipids between the ER and plasma membrane bilayers via a unique lipid-harboring domain [ 44 ]. Table 3 Membrane contact site (MCS) proteins enriched in cardiac ER/SR MCS proteins are sorted by A SR values, and their rank order among 354 proteins that are in SERCA-positive SR (Rank). High values of A SR for VAP-B and VAP-A reflect the importance of cardiac ER/SR in transporting proteins and lipids to distal parts of the myocyte. Other proteins involved in ER inter-organellar contact include junctophilin-2, and extended synaptotagmins. Only junctophilin-2 showed a negative E sub value, consistent with its reported enrichment in junctional SR (cf. Figure 3 A). Membrane contact site protein Rank A SR E SR E sub Role VAP-B (vesicle-associated membrane protein (VAMP)-associated protein B) 10 18.5 5.6 0.40 Associated with VAMP subfamily of SNARES; involved in lipid transfer from ER on lipid transfer proteins. VAP-A 24 11.3 7.4 0.29 junctophilin-2 55 5.9 3.1 -0.23 Binds to PI enriched plasma membrane lipids (MORN sites) at junctional SR. extended synaptotagman-2 288 0.55 5.7 0.33 Tethers ER to the plasma membrane by C-terminal MCS, and transports glycerolipids between the two bilayers. extended synaptotagman-1 308 0.37 3.1 0.13 Also yielding a high A SR value was fat storage-inducing transmembrane protein 2 (FIT2) ( A SR = 18.5, 11th highest ), which plays an important role in the formation of lipid droplets and the distribution of lipids between the ER membrane and lipid droplets [ 45 – 47 ]. Alpha crystalline B ( A SR = 18.3, 12th highest ) occurs in situ as globular polymers [ 48 ]. In total, five prominent filamentous proteins co-enriched with SERCA-positive membrane: desmin and crystalline αB, vimentin 12, keratin II 6B, and beta-tubulin 2C (Table 4, Appendix ). 5. Functional sets of proteins in cardiac ER/SR The identification of sets of proteins related to known SR and ER functions, added consistency and context to the 354 proteins found in our analysis. Proteins involved in related ER/SR functions were present as complete sets in SERCA-positive membranes. And, while these ER/SR subdomains were present in widely varying abundances (A SR values), their enrichment factors (E SR and E sub ) were remarkably similar, consistent with their distribution together in membrane patches (vesicles after homogenization). The vast majority of these 354 proteins have not been previously identified in cardiac SR. We briefly summarize the major functional sets of proteins found, and compare their constituent protein enrichments and relative abundances. 5.1 SR Ca pumping The most abundant peptide spectra, not surprisingly, were from SERCA2. Peptides from phospholamban, a subunit of SERCA2a that dissociates with PKA-dependent phosphorylation were detected at roughly 50% of SERCA2. The need for inclusion of the MW factor to the calculation of A SR values can be easily appreciated in this case, with the 20-fold difference in mass between SERCA and phospholamban molecules. While the relative levels of SERCA2 and phospholamban from this single mongrel canine heart sample will be subject to error and biological variability, the set of three proteins that function in Ca pumping (SERCA2, phospholamban, and SERCA1) were the 1st, 4th, and 15th highest ASR values, accounting for roughly 11% of spectra, supporting the view that Ca accumulation is a primary function of the cardiac ER/SR. Their enrichments in SR (E SR ), and distribution between MedSR and HighSR membrane subcompartments (E sub ) were also highly similar (Fig. 5 and Online Resource S1). 5.2 Intraluminal ER/SR proteins Intraluminal proteins represented an abundant set of proteins based on their number and high A SR values, constituting about 12% of total SR protein, with about half of the mass due to sarcalumenin and calsequestrin (Table 5 ). Many luminal SR proteins are those carrying a C-terminal sequence Lys-Asp-Glu-Leu (KDEL), which interacts with KDEL receptors to retrieve these proteins from the Golgi back to ER compartments [ 49 – 52 ]. Protein disulfide isomerase (PDI) isoforms, although not previously identified in heart tissue, were among the proteins most enriched in SR (Table 5 ); also, more highly enriched in HighSR than MedSR (Fig. 3 A) consistent with the common notion that free SR is essentially cardiac smooth ER, and that it contains less RyR [ 53 ]. 5.3 Junctional SR proteins The Ca oxalate loading method was previously used to identify several major junctional SR proteins, including calsequestrin-2, cardiac triadin, junctin, and RyR2 [ 15 , 16 , 54 , 55 ]. These 4 major junctional SR proteins, along with junctophilin-2, all presented here with relatively high levels of A SR , accounting for roughly 5% of total SR peptide spectra. Levels of couplon proteins reported in rabbit fast-twitch skeletal muscle SR [ 56 ] show interesting parallels (Fig. 6 ). Junctin, a splice variant of junctate and aspartyl β-hydroxylase [ 57 ], could not be distinguished based on the peptides sequenced. Treves et al. [ 58 ] previously reported that no gross differences occurred immunologically between junctin and junctate levels in heart homogenates, suggesting that junctin may be roughly half of the A SR level reported here. Co-enrichment of L-type Ca channel with junctional SR markers was further evidence of a stable protein complex of couplon proteins with the sarcolemmal T-tubule membrane [ 21 ]. Table 5 Proteins of the ER/SR lumen Resident luminal ER/SR proteins, listed in order of decreasing A SR . Proteins known to be highly specific for cardiac and skeletal muscle myocytes cells are indicated ( shaded rows ) [ 18 ]. All other proteins likely act as ER protein chaperones, and are maintained in ER through a C-terminal –KDEL retrieval signal ( asterisk *). Calnexin is a Type I transmembrane protein with a large luminal segment that has calreticulin-like chaperone activity [ 59 ]. GRP78, glucose-regulated protein, M r = 78 kDa; PDI, protein disulfide isomerase; His-rich Ca BP, histidine-rich Ca binding protein. Rank identifies proteins by their order among 354 proteins in SERCA-positive SR sorted by A SR (see Online Resource 1). Protein KDEL Rank A SR E SR E sub sarcalumenin 3 73.2 4.6 0.42 calsequestrin 5 41.1 4.7 − 0.15 GRP78 * 13 17.3 5.0 0.49 PDI A3 * 32 9.6 6.2 0.36 GRP94 * 36 8.5 5.7 0.54 calnexin 41 7.7 6.8 0.36 PDI A2 * 73 4.9 5.1 0.46 PDI A4 * 91 4.3 7.8 0.40 PDI A6 * 94 4.2 15.2 0.59 calreticulin * 142 2.4 8.5 0.54 PDI * 145 2.3 7.0 0.41 PDI isoform TMX * 159 2.1 6.8 0.11 His-rich Ca BP 217 1.2 5.0 0.19 5.4 Peroxisomal proteins and rab proteins in MedSR In addition to the enrichment of junctional SR proteins, MedSR membranes were also enriched in peroxisomal proteins, rab proteins, and caveolar proteins (Fig. 7 ), but the three types of protein-containing vesicles varied in their membrane enrichments. Peroxisomal proteins were very uniquely enriched in SERCA-positive membranes, and their enrichment was highly variable (E SR =26.7 ±22.3) (Fig. 8A). In addition, peroxisomal proteins were extraordinarily enriched in MedSR compared to HighSR membranes (avg E sub = -0.50 ±0.09). By comparison, junctional SR proteins were much less enriched in MedSR compared to HighSR (avg E sub = -0.24 ±0.10, for 4 couplon proteins). The reason for this unusual enrichment of peroxisomal proteins in SERCA-positive membranes is unknown, and much remains to be learned about this peculiar ER subdomain [ 60 – 62 ]. When E sub values were averaged for several sets of functionally related proteins, average E sub values covered a range of distribution between the two SR subpopulations (Fig. 8A), suggesting that individual membrane patches are enriched in separate functional subdomains, with each subdomain exhibiting particular Ca transport properties that reflect its inclusion of SERCA and RyR protein. Rabs and other small GTPases were also highly enriched in MedSR, exhibiting E sub values similar to those of known junctional SR proteins (Fig. 8A, B). Rab proteins are fundamental regulators of organelle biogenesis and vesicle transport, and constitute the largest subset of the ras superfamily of small GTPases [ 63 ]. In addition to abundant rabs, the rab associated protein PRA-1 (A SR = 25.0, 7th highest ), and hedgehog acyltransferase-like protein (aka mitsugamin-56 [ 64 ]) were also enriched in SR (Online Resource 1). The possible close physical proximity of rabs to junctional SR sites is consistent with evidence of protein secretion in cultured cardiomyocytes emanating from sites close to junctional SR [ 65 ]. 5.5 Rough ER proteins - Cardiac rough ER is a critical subdomain of ER, and in cardiomyocytes has a predominantly perinuclear morphology that is distinct from the repeating SR sarcomeres that control contraction [ 65 , 66 ]. Yet, SERCA-positive SR membrane vesicles also contained a complete collection of known rough ER proteins involved in translation, translocation, and N-linked glycosylation (Table 6, Appendix ). Rough ER proteins were of relatively low abundance (A SR = 4.0 ± 1.7), but very highly-enriched over crude MVs (E SR = 6.0 ± 2.5) (Fig. 8A). 5.6 Lipid metabolism and lipid modifications of proteins - Numerous enzymes involved in lipid metabolism were enriched in SERCA-positive membranes. The highest A SR value (= 20.7) resulted from CDITP, which appends inositol-3-phosphate to diacylglycerol, with numerous lipid metabolizing proteins present at lower A SR levels (Fig. 9, Appendix ). 5.7 Proteins involved in ER membrane structure and dynamics - Many cardiac ER/SR proteins are those thought to play roles in distributing and trafficking proteins across the biosynthetic pathway. Several act by guiding membrane patches along transport filaments; these include Ca-binding protein p22, vesicle-trafficking protein Sec22b, vesicle-associated membrane protein 2 (VAMP-2), and vesicle transport protein Sec20 [ 67 – 70 ]. The mammalian proteins of the p24 family (TMED 10, 9, 2, 1) are involved in selective loading of cargo in transport vesicle between membrane compartments, and the co-enrichment and relative abundances of its known subunits support a role in cardiac ER/SR protein distribution [ 71 , 72 ]. Finally, other ER proteins may function in the maintaining the structure of ER subcompartments, such as reticulons-2 and − 4 [ 73 ], lunapark-3 [ 74 ], and climp-63 [ 75 ]. Proteins involved in ER/SR dynamics are tabulated, along with enrichment values, in Table 7, Appendix . Conclusions In this study, we used the technique of Ca oxalate loading of cardiac SR membrane vesicles to produce two SR membrane subpopulations (MedSR and HighSR), thereby enriching patches of cardiac ER/SR membrane that contain combinations of SERCA and RyR levels. Roughly one third of microsomal proteins were enriched in SERCA-positive membranes, and about a third of those were more enriched in the MedSR membranes, indicating the presence of enriched RyR in the same SR patch, and suggesting a relative proximity to junctional SR, or at least a biochemically distinct membrane subdomain. The activation of SERCA activity in our current study, an historical measure of SR function, led to the enrichment of proteins from every ER subcompartment, supporting a view that cardiac ER and SR should cannot be demarcated based only upon their ability to function in Ca handling. Proteins enriched in SERCA-positive SR vesicles were defined as any protein that was enriched ≥2.0-fold over its level in crude heart microsomes. This single enrichment criterion selected 354 proteins of 1102 total proteins in crude microsomes, while excluding all mitochondrial, contractile protein, and other known organellar contaminants. Even major mitochondrial and contractile protein contaminants were eliminated by this simple measure of enrichment. SERCA-positive SR proteins encompassed proteins from all known functional ER and SR subdomains, leading us to conclude that SERCA-positive membranes represent cardiac ER and SR. Plotting A SR values for the top 2–4 proteins of different groups of proteins (Fig. 10 ), provided some semi-quantitative insight into how different ER/SR subdomains may contribute to overall SR function. In spite of these substantial variations in A SR among functional sets of proteins, the enrichment properties E sub and E SR among the same sets were remarkably consistent; both because of their physical segregation in membrane vesicles, but also because enrichment values are derived from ratios of A SR values in two preparations. For example, a large number of known KDEL proteins were found in SR, exhibiting a wide range of A SR values (6.3 ±9.0, N = 7 proteins). For the same 7 KDEL proteins, however, E sub values were 0.47 ±0.08, and E SR values were 7.1 ±3.7. The enrichment of junctional SR proteins in MedSR membranes was previously demonstrated by immunoblot analyses to be a feature of this SR preparation [ 5 , 15 , 18 , 21 , 76 ]. In the present study, we found that the values of E sub for 354 proteins formed a continuous range from − 0.9 (largely detected only in MedSR) to + 0.9 (largely detected only in HighSR membranes) (Fig. 4 ). Other functional protein groups exhibited similarly segregated values (Figs. 3 , 4 ,8), suggesting that they too were physically distributed into at least two divergent subcellular sites: those closer to junctional SR and those further removed from such sites. The segregation of protein functional groups in terms of E SR and E sub shows that vesicles are derived from small enough membrane surfaces to fractionate with different enrichment patterns, and do not simply represent huge sections of membrane surface. In summary, enrichment of cardiac membranes by Ca oxalate loading leads to the enrichment of ER/SR proteins, distributed between membrane fractions that differ in Ca leak through RyR. The distribution of individual proteins between the two fractions (Esub), and the consistent enrichments found among different functional sets of proteins, reflects the connections between ER/SR subdomains and the well-studied spatial relationships between junctional and free SR. Our data present for the first time a reliable estimation of cardiac ER/SR protein content, along with a semi-quantitative assessment of prominent sets of functional ER/SR subdomains present in a microsomal preparation of canine ventricular tissue. Declarations Funding We acknowledge the support of a grant from the Office of the Vice President for Research at Wayne State University (S.C.). Assistance of the Wayne State University Proteomics Core was supported through NIH grants P30 ES020957, P30 CA 022453 and S10 OD010700. This work was also supported by the National Institutes of Health [1R01DK110314 (X. C.)], and the American Heart Association. [18TPA34170284 /ZC/2018]. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Steven Cala: Conceptualization, Methodology, Writing. Nicholas Carruthers: Data curation, Software . Paul Stemmer: Supervision, Investigation, Methodology. Zhenhui Chen: Validation, Investigation. Xuequn Chen: Resources, Conceptualization. Data Availability The datasets generated during and/or analyzed during the current study are available at Figshare.com; Filename: CardiacSRproteome; DOI: 10.6084/m9.figshare.19953701. Raw data and search results have been submitted to the ProteomeXchange via PRIDE with accession PXD022455 (Username: [email protected] , Password: G1wlk66N). Ethics approval The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animal research was approved by the Wayne State University Animal Investigation Committee (protocol #A 04-02-13). References Bennett HS and Porter KR (1953) An electron microscope study of sectioned breast muscle of the domestic fowl. Am J Anat 93:61-105. doi: 10.1002/aja.1000930104 Porter KR and Palade GE (1957) Studies on the endoplasmic reticulum. III. Its form and distribution in striated muscle cells. 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Supplementary Files Appendix2523.docx Cite Share Download PDF Status: Published Journal Publication published 10 Apr, 2023 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Major revision 19 Feb, 2023 Reviews received at journal 14 Feb, 2023 Reviewers agreed at journal 07 Feb, 2023 Reviewers invited by journal 07 Feb, 2023 Editor assigned by journal 07 Feb, 2023 Submission checks completed at journal 07 Feb, 2023 First submitted to journal 06 Feb, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Cala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYLCCBww2MCYzkVoSGNJI13KYBC3yDcwPPyTUnE/sn5F78AFDhXViAyEtBgfYjCUSjt1OnHEjL9mA4Uw6EVoYeBgkEthu5zbcyDGTYGw7TFiLfAMP84+Ef+dy59/IMf/B+I8ILQwHeNgkEtsO5G4A2sLA2ECEFoPDbGYWiX3J9RvPvAF5Kt2YsMPamx/f+PDNzljueI7hhw811rKEHQaPCIEEUASRBPgPkKZ+FIyCUTAKRg4AAOU4P776wL+qAAAAAElFTkSuQmCC","orcid":"","institution":"Wayne State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Steven","middleName":"E.","lastName":"Cala","suffix":""},{"id":173999306,"identity":"9705a658-df05-4a4b-baa6-0e83cb89ab74","order_by":1,"name":"Nicholas J. 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Even when visualized by protein stain, one sees proteins more enriched in MedSR and HighSR (\u003cem\u003earrowhead\u003c/em\u003e) or less enriched (\u003cem\u003edouble arrowheads\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/e29cd6ad667ed221ad6ac666.png"},{"id":32662010,"identity":"fc587d3f-0ff0-40c3-b7d0-ce849108ea0b","added_by":"auto","created_at":"2023-02-08 18:22:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":219258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein enrichments resulting from Ca oxalate loading of SR vesicles.\u0026nbsp; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e, sorting of proteins identified in crude cardiac microsomal vesicles (MV) in order of decreasing peptide abundance, with only the first 200 proteins shown here (\u003cem\u003eblue bars\u003c/em\u003e)\u003cem\u003e. \u003c/em\u003eSuperimposed is a plot of their fold enrichment in SR fractions (\u003cem\u003ered bars\u003c/em\u003e). Only about 40 of the 200 were enriched by SERCA activation (E\u003csub\u003eSR \u003c/sub\u003e= 2.0, \u003cem\u003epale gray line\u003c/em\u003e), defining a set of SR proteins, as discussed in this study. The most abundant SR-enriched protein was SERCA2.\u0026nbsp; \u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e, sorting of all 354 SR-enriched proteins by E\u003csub\u003eSR \u003c/sub\u003evalues (which includes the 40 red values in panel A). Shown in green are the E\u003csub\u003eSR\u003c/sub\u003e values of 14 known cardiac SR proteins, enriched in SERCA-positive membranes on an average of 5.0 ±1.6 (SD) over crude membrane vesicles; 2.0-fold enrichment in SR is marked (\u003cem\u003earrowhead\u003c/em\u003e). CXN, \u003cem\u003ecalnexin\u003c/em\u003e; JCT, \u003cem\u003ejunctin plus junctate\u003c/em\u003e; PLB, \u003cem\u003ephospholamban\u003c/em\u003e; CSQ2, \u003cem\u003ecalsequestrin-2\u003c/em\u003e; TRD, \u003cem\u003etriadin\u003c/em\u003e; HRC, \u003cem\u003ehistidine-rich Ca-binding protein\u003c/em\u003e; SCL, \u003cem\u003esarcalumenin\u003c/em\u003e; JP2, \u003cem\u003ejunctophilin-2\u003c/em\u003e; glucose regulated protein of M\u003csub\u003er\u003c/sub\u003e=94,000, GRP94\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/0a1220f0a5eefacaaac70cbf.png"},{"id":32661730,"identity":"c5706449-3cb2-43f4-98cc-bfdd6ccea55e","added_by":"auto","created_at":"2023-02-08 18:14:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnown cardiac SR proteins distribute between HighSR and MedSR consistent with previous immunological studies.\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003e\u0026nbsp;A\u003c/strong\u003e\u003c/em\u003e, well-studied SR proteins exhibit E\u003csub\u003esub\u003c/sub\u003e values from +0.60 to -0.45 (\u003cem\u003eleft hand y-axis\u003c/em\u003e), which corresponds to a roughly 4-fold greater level in HighSR membranes to 3-fold greater in MedSR, respectively (\u003cem\u003eright hand y-axis\u003c/em\u003e). Negative values of E\u003csub\u003esub\u003c/sub\u003e based on mass spectrometry data, predict the same SR subdomain distribution described in the literature for 15 non-junctional SR (\u003cem\u003eblue bars\u003c/em\u003e) and 7 junctional SR (\u003cem\u003ered bars\u003c/em\u003e) proteins, based on immunological studies. Three SR proteins involved in Ca (oxalate) uptake (\u003cem\u003ewhite bars\u003c/em\u003e) were more evenly distributed between SR subfractions. \u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e, Relative abundances for the same 22 proteins show far greater variability. The A\u003csub\u003eSR\u003c/sub\u003e value for junctin/junctate (JCT/JCTA) is shaded in two halves (\u003cem\u003easterisk\u003c/em\u003e, *) to approximate the portion that corresponds only to junctin. PDI, \u003cem\u003eprotein disulfide isomerase\u003c/em\u003e; Calret, \u003cem\u003ecalreticulin; \u003c/em\u003eGRP94, \u003cem\u003eglucose regulated protein of M\u003c/em\u003e\u003csub\u003e\u003cem\u003er\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e=94,000\u003c/em\u003e; SCL, \u003cem\u003esarcalumenin\u003c/em\u003e; Calnex, \u003cem\u003ecalnexin\u003c/em\u003e; HRC, \u003cem\u003ehistidine-rich Ca-binding protein\u003c/em\u003e; PLB, \u003cem\u003ephospholamban\u003c/em\u003e; CSQ2, \u003cem\u003ecalsequestrin-2;\u003c/em\u003e JP2, \u003cem\u003ejunctophilin-2\u003c/em\u003e; TRD, \u003cem\u003etriadin\u003c/em\u003e; LTCC\u003cem\u003e, L-type Ca channel a-subunit.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/947614a3afe97db5fe9d8fb1.png"},{"id":32661343,"identity":"9bda23f3-87ba-496d-ba72-59807355e022","added_by":"auto","created_at":"2023-02-08 18:06:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistributions between HighSR and MedSR are similar for sets of proteins with similar functions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE\u003csub\u003esub\u003c/sub\u003e values for all 354 SERCA-positive SR proteins (\u003cem\u003eshaded gray area\u003c/em\u003e) range from +0.90 to -0.90 (90% more enriched in HighSR or in MedSR, respectively). Distribution of any one protein between the two density layers reflects in part, its physical proximity to junctional SR sites. Co-enrichment of proteins with similar function within common membrane fragments therefore reflect similar relative levels of (nearby) SERCA2 and RyR.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/b53f656289fa1c6c77c42cc3.png"},{"id":32661347,"identity":"583890eb-8296-4f45-ad9f-335bc169ca45","added_by":"auto","created_at":"2023-02-08 18:06:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 15 most abundant SR proteins based on A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eSR\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e values.\u003c/strong\u003e \u0026nbsp;A\u003csub\u003eSR\u003c/sub\u003e values are expressed as a percentage of SERCA2 A\u003csub\u003eSR\u003c/sub\u003e (=100.0). An asterisk denotes the fact that junctin and junctate were not distinguishable in this study. The 15 proteins shown represented 38% of all spectra from all 354 proteins in SERCA-positive membranes.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/fd2ead99feee1afc44bb1ad8.png"},{"id":32661731,"identity":"9878ee58-7824-4dec-b295-4bfc6af04276","added_by":"auto","created_at":"2023-02-08 18:14:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eSR\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e values for well known junctional SR proteins from a preparation of cardiac or skeletal muscle SR.\u003c/strong\u003e\u0026nbsp; Levels of individual proteins are normalized to that of either SERCA2a (=100.0) for cardiac (Card) SR, or to the levels of SERCA1 (=100.0) for rabbit fast-twitch muscle (Skel) SR proteins as reported in ref. [56]. Note differences in ordinate scales for the three panels shown. Skel data was obtained from a similar proteomic analysis by Liu et al. [56] using the classic preparation of heavy SR prepared from rabbit fast-twitch skeletal muscle that is based upon microsome sedimentation rates [8]. RyR, ryanodine receptor; CSQ, calsequestrin-2; JCT, junctin (plus junctate for Card data); TRD, triadin (cardiac isoform for Card); JP, junctophilin; LTCC, L-type Ca channel. Text below bars designates isoforms or subunits. Double shading of Card JCT denotes the approximate half of peptide spectra that may have arisen from junctate.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/3339db37c3aae91e8a2a8e9d.png"},{"id":32661735,"identity":"e5e70ad9-7289-4c23-b8d5-aa41954f4ddc","added_by":"auto","created_at":"2023-02-08 18:14:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":104084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJunctional SR and other ER/SR subdomains enriched in MedSR membranes.\u0026nbsp; \u003c/strong\u003eSR proteins with negative E\u003csub\u003esub\u003c/sub\u003e values (more enriched in MedSR) were sorted by A\u003csub\u003eSR\u003c/sub\u003e (highest to lowest).\u0026nbsp; Only the 50 highest A\u003csub\u003eSR\u003c/sub\u003e values are shown.\u0026nbsp; Rab isoforms were an abundant set of enriched proteins (\u003cem\u003egreen bars with labeled isoform\u003c/em\u003e). The total peptide spectra for JCT plus junctate is shown here (JCT, \u003cem\u003easterisk \u003c/em\u003e*).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/867245350979f4ca9508177d.png"},{"id":32661733,"identity":"91d7dd7c-9564-4415-a9a0-5d0722294b12","added_by":"auto","created_at":"2023-02-08 18:14:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":79064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSets of functionally related proteins undergo similar enrichments \u003c/strong\u003e\u0026nbsp;\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e, Subsets of ER/SR proteins with related functions: average enrichment in SR (E\u003csub\u003eSR\u003c/sub\u003e mean ± S.D., \u003cem\u003ey-axis\u003c/em\u003e) and average distribution between SR subfractions (E\u003csub\u003esub\u003c/sub\u003e mean ± S.D.), \u003cem\u003ex-axis\u003c/em\u003e. The numbers shown in parentheses are the number of proteins averaged from each set. \u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e, E\u003csub\u003eSR\u003c/sub\u003e and E\u003csub\u003esub\u003c/sub\u003e values for individual rabs (\u003cem\u003epurple rings\u003c/em\u003e) and other small GTPases are shown.\u0026nbsp; Abundance values (A\u003csub\u003eSR\u003c/sub\u003e) are proportional to symbol diameters. The 3 most abundant rabs are labeled (2A, 1A, and 1B).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/8945388eb7dddc3031f9777f.png"},{"id":32661353,"identity":"7b5a0ba0-0098-4b23-aa2f-21cda636700f","added_by":"auto","created_at":"2023-02-08 18:06:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":161677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 2-4 most abundant ER/SR proteins in each functional subset.\u003c/strong\u003e\u0026nbsp; A\u003csub\u003eSR\u003c/sub\u003e values sharply decline for the first 2-4 protein of each functional set. Plots are offset along the x-axis to improve visual clarity. A\u003csub\u003eSR\u003c/sub\u003e values for junctional SR proteins only include transmembrane proteins (cf. Fig. 6), excluding calsequestrin-2 which is grouped with the very abundant sarcalumenin as two very abundant luminal proteins of uncertain function.\u0026nbsp; Proteins with common functions are represented in color-coded sets using similar color tags. Proteins are also color tagged in the table of all proteins (Online Resource 1).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/08d0f59ab15897915db6ead7.png"},{"id":44724752,"identity":"73955d1f-76c8-4928-bb2f-77a4923d90ba","added_by":"auto","created_at":"2023-10-16 20:34:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1953429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/c5a288ea-e503-4f40-b29d-595e1f056293.pdf"},{"id":32661355,"identity":"05802d4e-4b2f-416c-ba29-88caec32a871","added_by":"auto","created_at":"2023-02-08 18:06:29","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":250104,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix2523.docx","url":"https://assets-eu.researchsquare.com/files/rs-2557992/v1/43168f7b411728683b41cdae.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Activation of Ca transport in cardiac microsomes enriches functional sets of ER and SR proteins","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe basic morphology of the endoplasmic reticulum (ER) in muscle has been known since the initial electron microscopic studies in the 1950\u0026rsquo;s by Bennett and Porter [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and Palade and Porter [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], when it was termed the sarcoplasmic reticulum (SR)\u003csup\u003e1\u003c/sup\u003e, given its unique structure that repeats across all sarcomeres in the myocyte. Studies over many decades have focused on its Ca handling properties, beginning with extensive characterization of the important Ca translocating ATPase activity, now termed SERCA, for SR, ER-Ca-ATPase [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Much later, a ryanodine-sensitive Ca-releasing activity was identified [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Extensive analyses of Ca transport in membrane preparations from skeletal muscle also led to established membrane subfractionation protocols, and isolation of membranes of distinct densities that contained different protein composition. Meissner [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] reported the first fractionation of skeletal muscle membranes by isolating a fraction of heavy vesicles that contained the electron-opaque protein polymer calsequestrin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and later shown to contain foot processes consistent with morphological depictions of junctional SR [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The lighter membranes showed less of the dense luminal protein content, and was concluded to contain free (non-junctional) SR vesicles [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA technique for subfractionation of microsomes from heart tissue was later developed that took advantage of the ability of cardiac SR microsomes to concentrate Ca oxalate within their lumens [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While these studies were originally developed to biochemically separate sarcolemma and SR, subsequent work [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] showed that the Ca oxalate loading had actually produced two distinct types of SR membranes, with roughly half of membrane protein in the densest membrane fraction. And though it was known by then that the drug ryanodine could stimulate Ca accumulation into SR membrane vesicles at high concentrations, these dense membranes were ryanodine insensitive [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, a second membrane fraction of lower density (unable to traverse a 1.5 M sucrose cushion) was highly sensitive to ryanodine [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Indeed, by inhibiting Ca leak, 0.3 mM ryanodine added during membrane isolation converted SR membranes of lower density into high density (high Ca oxalate) vesicles [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The densities of SR membrane vesicles, as well as the distribution of SR proteins between the two subfractions, are regulated by the relative levels of SERCA and ryanodine receptor (RyR) contained in the fragmented SR patches.\u003c/p\u003e \u003cp\u003eSeveral studies have used SERCA-positive membrane subpopulations to either validate the identity of a putative junctional SR protein [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], or non-junctional SR protein [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this study, we determined the complete protein compositions of membrane vesicles generated by SERCA-depending Ca oxalate loading. We found 354 proteins that were enriched as a result of SERCA activation. In addition, a co-enrichment of functionally related proteins resulted from their similar distribution among membrane patches, and reflect the enrichment of ER/SR subdomains in close proximity to SERCA2 and RyR2 activities in intact cardiomyocytes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cem\u003ePreparation of Crude Cardiac Sarcoplasmic Reticulum Vesicles\u003c/em\u003e - Cardiac microsomes were isolated from female mongrel dog left ventricular tissue, and loaded with Ca oxalate as previously described, with minor modifications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, heart tissue was homogenized in 10 mM NaCO\u003csub\u003e3\u003c/sub\u003e at 1:20 dilution (buffer volume/tissue wet weight). Cardiac microsomes were isolated by differential centrifugation, isolating microsomes between 10,000\u0026ndash;75,000 x g\u003csub\u003emax\u003c/sub\u003e. In contrast to the previously published method, a final wash of the pellet in 0.6 M KC1, 30 mM histidine, pH 7.0, was not carried out. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). Animal research was approved by the Wayne State University Animal Investigation Committee (protocol #A 04-02-13).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCa oxalate loading of SR -\u003c/em\u003e Loading of crude cardiac microsomes, 75 mg of protein were resuspended in 40 ml of an ice-cold medium containing 50 mM histidine, 100 mM KCI, 65 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 60 mM Na\u003csub\u003e2\u003c/sub\u003eATP, 25 mM Tris/EGTA, 20 mM CaCl\u003csub\u003e2\u003c/sub\u003e, and 5 mM Tris oxalate (pH 7.1). The suspension was rapidly warmed to 37 \u003csup\u003eo\u003c/sup\u003eC to initiate active and rapid Ca uptake, and the incubation was conducted for 10 min with 5 mM additional Tris/oxalate added after the first 5 min of incubation. The suspension was then immediately centrifuged at 4 \u0026ordm;C for 30 min at 100,000 x g\u003csub\u003emax\u003c/sub\u003e. The resulting brownish membrane pellet included a central white region, indicative of the Ca oxalate precipitate inside the highest density membranes. The whitish center was not present if ATP was not included in the Ca oxalate loading step (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIsolation of medium and high density membrane vesicles after Ca oxalate loading\u003c/em\u003e \u0026ndash; Subfractionation of Ca loaded membranes was carried out essentially as previously described [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, membrane pellets were resuspended in 0.25 M sucrose containing 300 mM KC1, 50 mM sodium pyrophosphate, and 100 mM Tris (pH 7.2). This material was layered over a discontinuous sucrose gradient of 0.6 M, 0.8 M, 1.0 M, and 1.5 M sucrose dissolved in the same buffer. After centrifugation at 125,000 x g\u003csub\u003emax\u003c/sub\u003e for 2 h, membranes were collected from both the 1.5 M sucrose cushion (MedSR) or as the white pellet at the bottom of the gradient (HighSR). These two subfractions were not present in membranes not Ca loaded (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). MedSR was diluted with 4 volumes of ice-cold H\u003csub\u003e2\u003c/sub\u003eO, and then sedimented at 105,000 x g\u003csub\u003emax\u003c/sub\u003e for 60 min. MedSR and HighSR were resuspended in 0.25 M sucrose, 10 mM histidine, and stored frozen at -20 \u003csup\u003eo\u003c/sup\u003eC. Protein was determined by the method of Lowry et al. (17).\u003c/p\u003e\u003cp\u003e \u003cem\u003ePreparation of protein samples for mass spectrometric analysis\u003c/em\u003e - To evaluate the protein composition of the two SERCA-positive membrane fractions, relative to the crude cardiac membranes, we analyzed exactly 20 \u0026micro;g each of MVs, MedSR, and HighSR membranes by SDS-PAGE. The wet gel was stained with SPYRO Ruby red to visualize the protein separations qualitatively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cem\u003eTryptic digestion of SR samples\u003c/em\u003e - For in-gel digestion (GeLC-MSMS), each lane (crude cardiac microsomes (MV), MedSR, or HighSR membranes) was cut into 20 gel slices for improved sampling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Gel slices were equilibrated in H2O, dried, and digested with trypsin added in dilute solution (1:10 w/w) and digested overnight at 37 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLC-MS/MS analysis and database search\u003c/em\u003e - Tryptic peptides were separated by reverse phase chromatography (Magic C18 column, Michrom), followed by ionization with the ADVANCE ion source (Michrom), and then analyzed in an LTQ-XL mass spectrometer (Thermo Fisher Scientific). For each MS scan, up to seven MS/MS scans were obtained using collision-induced dissociation. Data analysis was performed using Proteome Discoverer 1.1 (Thermo) which incorporated the Mascot algorithm (Matrix Science). The NCBI database was used against mammalian protein sequences and a reverse decoy protein database was run simultaneously for false discovery rate (FDR) determination. Secondary analysis was performed using Scaffold (Proteome Software). A fixed modification of +\u0026thinsp;57 on cysteine (carbamidomethylation) and variable modifications of +\u0026thinsp;16 on methionine (oxidation) and +\u0026thinsp;42 on protein N-terminus (acetylation) were included in the database search. Minimum protein identification probability was set at \u0026ge;\u0026thinsp;95%, with 1 unique peptide at \u0026ge;\u0026thinsp;95% minimum peptide identification probability. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, along with their relative levels within each of the three different membrane fractions, when levels were sufficient. Raw data and search results have been submitted to the ProteomeXchange via PRIDE with accession PXD022455 (Username: [email protected], Password: G1wlk66N).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1. Protein composition of crude cardiac microsomes\u003c/h2\u003e \u003cp\u003eAn abundance \u003cem\u003eindex\u003c/em\u003e (A\u003csub\u003eSR\u003c/sub\u003e) was calculated for each protein, calculated as the number of its peptide spectra, divided by protein molecular weight as a rough correction for available target peptides, then normalized to the spectral level for SERCA2, which we defined as A\u003csub\u003eSR\u003c/sub\u003e=100.0. A\u003csub\u003eSR\u003c/sub\u003e values are a crude surrogate for protein abundance, since factors unrelated to protein abundance will affect the quantifcation of tryptic peptides. Nonetheless, A\u003csub\u003eSR\u003c/sub\u003e values across sets of functionally related proteins can provide useful comparisons.\u003c/p\u003e \u003cp\u003eIn a preparation of cardiac microsomes (MVs), we found 1102 proteins (see Online Resource 1). The most abundant peptides from crude cardiac MV membranes were from known mitochondrial proteins. The three proteins with the highest A\u003csub\u003eSR\u003c/sub\u003e values in cardiac microsomes were ATP/ADP translocase and ATP synthase, α and β subunits, with known mitochondrial proteins accounted for 7 of the top 10 proteins. The other three major proteins among the top ten highest A\u003csub\u003eSR\u003c/sub\u003e values were SERCA2, a keratin 1 isoform, and cardiac α1 actin. The focus of our experiments, however, was not directed at crude cardiac microsomal proteins, but instead, at proteins that are enriched in cardiac SR. Of the top 10 microsomal proteins detected, only SERCA2 was enriched in the denser membrane vesicles that result when Ca transport is activated by addition of ATP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2. Enrichment of membrane vesicles by SERCA activation\u003c/h2\u003e \u003cp\u003eThe two enriched SR subfractions analyzed in this study have previously been described as \u003cem\u003ejunctional SR\u003c/em\u003e and \u003cem\u003efree SR\u003c/em\u003e vesicles; however, with the much greater number of proteins identified through the use of our GeLC-MS/MS analysis, we have substituted the terms \u0026ldquo;medium-density SR membranes\u0026rdquo; (MedSR) and \u0026ldquo;high-density SR membranes\u0026rdquo; (HighSR), for these two membrane subpopulations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This terminology allows us to discuss the larger collection of proteins, whether or not they participate directly in Ca handling functions.\u003c/p\u003e \u003cp\u003eComparing the abundance of each protein (A\u003csub\u003eSR\u003c/sub\u003e) in MedSR and HighSR membranes with its abundance in MVs, we determined protein enrichments in SR (E\u003csub\u003eSR\u003c/sub\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Calculated from ratios of A\u003csub\u003eSR\u003c/sub\u003e values, the enrichment values were relatively unaffected by the sampling issues for any given protein. We used the value of E\u003csub\u003eSR\u003c/sub\u003e to define \u003cem\u003eSERCA-positive SR\u003c/em\u003e proteins as those proteins that were enriched by SERCA activation at least 2.0-fold (E\u003csub\u003eSR\u003c/sub\u003e \u0026ge; 2.0). For 354 SERCA-positive SR proteins, the enrichments varied, but the average enrichment was 8.0-fold (avg E\u003csub\u003eSR\u003c/sub\u003e = 8.0).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKey definitions and abbreviations used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eMembrane preparations\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCrude cardiac microsomal \u003cb\u003ev\u003c/b\u003eesicles\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMedium density Ca oxalate loaded SR, enriched in every junctional SR marker\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHighSR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHigh(est) density Ca oxalate loaded SR, enriched in free SR (non-junctional) vesicles\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eParameter determined for each protein\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eRange of values\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003eSR\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnrichment in SR \u003cem\u003e(MedSR\u0026thinsp;+\u0026thinsp;HighSR)/ MV\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ge; 2.0 for SR (average: 8.0-fold)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003esub\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnrichment in HighSR compared to MedSR\u003c/p\u003e \u003cp\u003e(\u003cem\u003eHighSR \u0026ndash; MedSR)/ (HighSR\u0026thinsp;+\u0026thinsp;MedSR)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e= 0.90 (90% higher in HighSR)\u003c/p\u003e \u003cp\u003e=\u0026thinsp;\u0026minus;0.90 (90% higher in MedSR)\u003c/p\u003e \u003cp\u003e= 0.00 (equally distributed)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003csub\u003eSR\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpectral Abundance in SR \u003cem\u003e(MedSR\u0026thinsp;+\u0026thinsp;HighSR)\u003c/em\u003e, divided by molecular weight, normalized to SERCA2 \u003cem\u003e(=\u0026thinsp;100.0)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u0026ndash;100.0\u003c/p\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e(SERCA2) \u0026equiv; 100.0\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn contrast to enrichment of SR proteins in total SR (HighSR \u003cem\u003eplus\u003c/em\u003e MedSR), enrichments in HighSR \u003cem\u003eversus\u003c/em\u003e MedSR membranes (E\u003csub\u003esub\u003c/sub\u003e), reflect differences in the way small membrane patches are pulled apart, yielding small vesicles with varying levels of Ca release activity (RyR) on top of their very active Ca accumulation. Thus, individual proteins that segregate with \u003cem\u003eMedSR\u003c/em\u003e were more likely to contain membrane patches that contain RyR; that is, closer to junctional SR sites \u003cem\u003ein vivo\u003c/em\u003e. To evaluate this distribution as a single number, we defined E\u003csub\u003esub\u003c/sub\u003e as the difference between levels in the two SR fractions divided by the total (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Proteins more enriched in High SR yielded a positive number with a theoretical maximal value of 1.0, and proteins enriched in MedSR yielded a negative number with a theoretical maximal value of -1.0. Values actually ranged from +\u0026thinsp;0.90 to -0.90.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3. Characterization of SR proteins based upon enrichments among membrane subpopulations\u003c/h2\u003e \u003cp\u003eOf the top 10 microsomal proteins detected in our ventricular muscle sample, only one (SERCA2) was enriched by SERCA activation (E\u003csub\u003eSR\u003c/sub\u003e=3.4). Among 200 microsomal proteins with the highest A\u003csub\u003eSR\u003c/sub\u003e values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cem\u003eblue bars\u003c/em\u003e), only 40 proteins were enriched at least 2.0-fold by SERCA activation (\u003cem\u003ered bars\u003c/em\u003e). All SR proteins previously identified as resident to cardiac SR using immunological methods were among the proteins enriched by SERCA activation in our study.\u003c/p\u003e \u003cp\u003eFor 14 of the best characterized SR proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cem\u003egreen bars\u003c/em\u003e), the average fold enrichment in SR over crude cardiac microsomes (E\u003csub\u003eSR\u003c/sub\u003e) was 5.0 \u0026plusmn;1.6 (mean \u0026plusmn;S.D.). When sorted based on their relative distribution between SR subcompartments (E\u003csub\u003esub\u003c/sub\u003e), all of the known junctional SR proteins were more enriched in MedSR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), consistent with the idea that MedSR membrane patches probably contain more RyR molecules, possibly combined with less SERCA. Luminal ER/SR proteins, on the other hand, were highly enriched in HighSR (except for calsequestrin-2). SERCA2, SERCA1, and phospholamban were more equally distributed among the membrane vesicles, consistent with a more equal activation of Ca loading in both SR populations. Despite the similar enrichments of known SR proteins, their relative abundances (A\u003csub\u003eSR\u003c/sub\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) varied greatly, consistent with data widely known from protein staining of SDS-gels [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the entire set of 354 proteins in SERCA-positive membranes, E\u003csub\u003esub\u003c/sub\u003e spanned a continuous range of values from +\u0026thinsp;0.90 to -0.90, corresponding to proteins at a 90% greater level in HighSR, or in MedSR, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Interestingly, for sets of proteins with related function, E\u003csub\u003esub\u003c/sub\u003e values were clustered together. 2, in support of the idea that membrane fragments are generated from ER/SR membrane patches that contain characteristic ratios of SERCA and RyR levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4. High abundance proteins illustrate wider functions in cardiac SR than only Ca handling\u003c/h2\u003e \u003cp\u003eA\u003csub\u003eSR\u003c/sub\u003e values are based upon the number of peptide spectra attributed to a particular protein, but are only semi-quantitative, as they assume similar coverage of every protein sequence by LC-MS/MS, which does not occur [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Yet, it was very interesting to look at the proteins with the highest A\u003csub\u003eSR\u003c/sub\u003e values, as it illustrates the scope of protein functions in cardiac SR. The 5 most abundant proteins in our SERCA-positive SR sample were SERCA2, desmin, sarcalumenin, phospholamban, and calsequestrin-2 (CSQ2), accounting for about a quarter of SR protein mass (percent of total spectra) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). SERCA2a and phospholamban are well known major constituents of cardiac SR [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and, as expected, their very high A\u003csub\u003eSR\u003c/sub\u003e values were a predictable consequence of their activity in Ca oxalate loading of SR vesicles. Sarcalumenin is a known luminal constituent of cardiac SR membranes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], but of uncertain function. It is comprised of two well known splice variants: one generally defined by its roughly 150-kDa apparent molecular weight on standard SDS-gels, and one defined by a roughly 53-kDa [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Desmin, a muscle-specific intermediate filament protein of striated muscle [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], was found at A\u003csub\u003eSR\u003c/sub\u003e levels comparable to SERCA2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSurprisingly high A\u003csub\u003eSR\u003c/sub\u003e values were also found for many proteins barely discussed in the cardiac research literature. For example, NADH-cytochrome b5 reductase gave the highest A\u003csub\u003eSR\u003c/sub\u003e value of a non-traditional cardiac SR protein (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 30.0; 6th highest)\u003c/em\u003e. Salviati et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] in 1981 reported very high levels of this redox enzyme, as well as cytochrome b5 itself (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 5.1; 69th highest)\u003c/em\u003e, in the SR of slow skeletal muscle. A prominent escort protein for rab proteins, PRA1 (prenylated rab acceptor 1), was prominently detected (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 25.0, 7th highest\u003c/em\u003e). The presence of abundant histone H4 (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 21.0, 8th highest\u003c/em\u003e), and other less abundant histones (Table\u0026nbsp;2, \u003cem\u003eAppendix\u003c/em\u003e) may suggest a role as a secreted antimicrobial protein [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] or copper reductase enzyme [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe high level of CDP-diacylglycerol-inositol 3-phosphatidyltransferase, isoform 1 \u003cem\u003e(CDITP, A\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 20.7, 9th highest)\u003c/em\u003e, was consistent with early studies by Kasinathan and Kirchberger [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Interestingly, the SR protein with the next highest A\u003csub\u003eSR\u003c/sub\u003e (VAP-B) is a prominent intra-organellar tethering protein that also plays a putative role in the transfer of synthesized PI to the plasma membrane. Both VAP-B and VAP-A \u003cem\u003e(A\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 18.5 and 11,3, 10th and 24th highest, resp.)\u003c/em\u003e were among multiple ER tethering proteins present [\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36 CR37 CR38\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The combined actions of these abundant ER/SR proteins may promote new plasma membrane sites for PI signaling activity in heart, as well as sites for junctophilin-2 binding and junctional SR formation [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Extended synaptotagmins, detected at low A\u003csub\u003eSR\u003c/sub\u003e levels, can also transport glycerolipids between the ER and plasma membrane bilayers via a unique lipid-harboring domain [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eMembrane contact site (MCS) proteins enriched in cardiac ER/SR\u003c/b\u003e MCS proteins are sorted by A\u003csub\u003eSR\u003c/sub\u003e values, and their rank order among 354 proteins that are in SERCA-positive SR (Rank). High values of A\u003csub\u003eSR\u003c/sub\u003e for VAP-B and VAP-A reflect the importance of cardiac ER/SR in transporting proteins and lipids to distal parts of the myocyte. Other proteins involved in ER inter-organellar contact include junctophilin-2, and extended synaptotagmins. Only junctophilin-2 showed a negative E\u003csub\u003esub\u003c/sub\u003e value, consistent with its reported enrichment in junctional SR (cf. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMembrane contact site protein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003csub\u003eSR\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE\u003csub\u003eSR\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE\u003csub\u003esub\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRole\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVAP-B (vesicle-associated membrane protein (VAMP)-associated protein B)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAssociated with VAMP subfamily of SNARES; involved in lipid transfer from ER on lipid transfer proteins.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVAP-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ejunctophilin-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBinds to PI enriched plasma membrane lipids (MORN sites) at junctional SR.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eextended synaptotagman-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTethers ER to the plasma membrane by C-terminal MCS, and transports glycerolipids between the two bilayers.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eextended synaptotagman-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e308\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlso yielding a high A\u003csub\u003eSR\u003c/sub\u003e value was fat storage-inducing transmembrane protein 2 (FIT2) (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 18.5, 11th highest\u003c/em\u003e), which plays an important role in the formation of lipid droplets and the distribution of lipids between the ER membrane and lipid droplets [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Alpha crystalline B (\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 18.3, 12th highest\u003c/em\u003e) occurs \u003cem\u003ein situ\u003c/em\u003e as globular polymers [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In total, five prominent filamentous proteins co-enriched with SERCA-positive membrane: desmin and crystalline αB, vimentin 12, keratin II 6B, and beta-tubulin 2C (Table\u0026nbsp;4, \u003cem\u003eAppendix\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e5. Functional sets of proteins in cardiac ER/SR\u003c/h2\u003e \u003cp\u003eThe identification of sets of proteins related to known SR and ER functions, added consistency and context to the 354 proteins found in our analysis. Proteins involved in related ER/SR functions were present as complete sets in SERCA-positive membranes. And, while these ER/SR subdomains were present in widely varying abundances (A\u003csub\u003eSR\u003c/sub\u003e values), their enrichment factors (E\u003csub\u003eSR\u003c/sub\u003e and E\u003csub\u003esub\u003c/sub\u003e) were remarkably similar, consistent with their distribution together in membrane patches (vesicles after homogenization). The vast majority of these 354 proteins have not been previously identified in cardiac SR. We briefly summarize the major functional sets of proteins found, and compare their constituent protein enrichments and relative abundances.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e5.1 SR Ca pumping\u003c/h2\u003e \u003cp\u003eThe most abundant peptide spectra, not surprisingly, were from SERCA2. Peptides from phospholamban, a subunit of SERCA2a that dissociates with PKA-dependent phosphorylation were detected at roughly 50% of SERCA2. The need for inclusion of the MW factor to the calculation of A\u003csub\u003eSR\u003c/sub\u003e values can be easily appreciated in this case, with the 20-fold difference in mass between SERCA and phospholamban molecules. While the relative levels of SERCA2 and phospholamban from this single mongrel canine heart sample will be subject to error and biological variability, the set of three proteins that function in Ca pumping (SERCA2, phospholamban, and SERCA1) were the 1st, 4th, and 15th highest ASR values, accounting for roughly 11% of spectra, supporting the view that Ca accumulation is a primary function of the cardiac ER/SR. Their enrichments in SR (E\u003csub\u003eSR\u003c/sub\u003e), and distribution between MedSR and HighSR membrane subcompartments (E\u003csub\u003esub\u003c/sub\u003e) were also highly similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Online Resource S1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e5.2 Intraluminal ER/SR proteins\u003c/h2\u003e \u003cp\u003eIntraluminal proteins represented an abundant set of proteins based on their number and high A\u003csub\u003eSR\u003c/sub\u003e values, constituting about 12% of total SR protein, with about half of the mass due to sarcalumenin and calsequestrin (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Many luminal SR proteins are those carrying a C-terminal sequence Lys-Asp-Glu-Leu (KDEL), which interacts with KDEL receptors to retrieve these proteins from the Golgi back to ER compartments [\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Protein disulfide isomerase (PDI) isoforms, although not previously identified in heart tissue, were among the proteins most enriched in SR (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e5\u003c/span\u003e); also, more highly enriched in HighSR than MedSR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) consistent with the common notion that free SR is essentially cardiac smooth ER, and that it contains less RyR [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e5.3 \u003cem\u003eJunctional SR proteins\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe Ca oxalate loading method was previously used to identify several major junctional SR proteins, including calsequestrin-2, cardiac triadin, junctin, and RyR2 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These 4 major junctional SR proteins, along with junctophilin-2, all presented here with relatively high levels of A\u003csub\u003eSR\u003c/sub\u003e, accounting for roughly 5% of total SR peptide spectra. Levels of couplon proteins reported in rabbit fast-twitch skeletal muscle SR [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] show interesting parallels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Junctin, a splice variant of junctate and aspartyl β-hydroxylase [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], could not be distinguished based on the peptides sequenced. Treves et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] previously reported that no gross differences occurred immunologically between junctin and junctate levels in heart homogenates, suggesting that junctin may be roughly half of the A\u003csub\u003eSR\u003c/sub\u003e level reported here. Co-enrichment of L-type Ca channel with junctional SR markers was further evidence of a stable protein complex of couplon proteins with the sarcolemmal T-tubule membrane [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eProteins of the ER/SR lumen\u003c/b\u003e Resident luminal ER/SR proteins, listed in order of decreasing A\u003csub\u003eSR\u003c/sub\u003e. Proteins known to be highly specific for cardiac and skeletal muscle myocytes cells are indicated (\u003cem\u003eshaded rows\u003c/em\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. All other proteins likely act as ER protein chaperones, and are maintained in ER through a C-terminal \u0026ndash;KDEL retrieval signal (\u003cem\u003easterisk\u003c/em\u003e*). Calnexin is a Type I transmembrane protein with a large luminal segment that has calreticulin-like chaperone activity [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. GRP78, glucose-regulated protein, M\u003csub\u003er\u003c/sub\u003e = 78 kDa; PDI, protein disulfide isomerase; His-rich Ca BP, histidine-rich Ca binding protein. Rank identifies proteins by their order among 354 proteins in SERCA-positive SR sorted by A\u003csub\u003eSR\u003c/sub\u003e (see Online Resource 1).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKDEL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRank\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003csub\u003eSR\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE\u003csub\u003eSR\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE\u003csub\u003esub\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esarcalumenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecalsequestrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGRP78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI A3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGRP94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecalnexin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI A4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI A6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecalreticulin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI isoform TMX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHis-rich Ca BP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e5.4 Peroxisomal proteins and rab proteins in MedSR\u003c/h2\u003e \u003cp\u003eIn addition to the enrichment of junctional SR proteins, MedSR membranes were also enriched in peroxisomal proteins, rab proteins, and caveolar proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), but the three types of protein-containing vesicles varied in their membrane enrichments. Peroxisomal proteins were very uniquely enriched in SERCA-positive membranes, and their enrichment was highly variable (E\u003csub\u003eSR\u003c/sub\u003e=26.7 \u0026plusmn;22.3) (Fig.\u0026nbsp;8A). In addition, peroxisomal proteins were extraordinarily enriched in MedSR compared to HighSR membranes (avg E\u003csub\u003esub\u003c/sub\u003e = -0.50 \u0026plusmn;0.09). By comparison, junctional SR proteins were much less enriched in MedSR compared to HighSR (avg E\u003csub\u003esub\u003c/sub\u003e = -0.24 \u0026plusmn;0.10, for 4 couplon proteins). The reason for this unusual enrichment of peroxisomal proteins in SERCA-positive membranes is unknown, and much remains to be learned about this peculiar ER subdomain [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen E\u003csub\u003esub\u003c/sub\u003e values were averaged for several sets of functionally related proteins, average E\u003csub\u003esub\u003c/sub\u003e values covered a range of distribution between the two SR subpopulations (Fig.\u0026nbsp;8A), suggesting that individual membrane patches are enriched in separate functional subdomains, with each subdomain exhibiting particular Ca transport properties that reflect its inclusion of SERCA and RyR protein.\u003c/p\u003e\u003cp\u003eRabs and other small GTPases were also highly enriched in MedSR, exhibiting E\u003csub\u003esub\u003c/sub\u003e values similar to those of known junctional SR proteins (Fig.\u0026nbsp;8A, B). Rab proteins are fundamental regulators of organelle biogenesis and vesicle transport, and constitute the largest subset of the ras superfamily of small GTPases [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In addition to abundant rabs, the rab associated protein PRA-1 \u003cem\u003e(A\u003c/em\u003e\u003csub\u003e\u003cem\u003eSR\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e= 25.0, 7th highest\u003c/em\u003e), and hedgehog acyltransferase-like protein (aka mitsugamin-56 [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]) were also enriched in SR (Online Resource 1). The possible close physical proximity of rabs to junctional SR sites is consistent with evidence of protein secretion in cultured cardiomyocytes emanating from sites close to junctional SR [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e5.5 \u003cem\u003eRough ER proteins\u003c/em\u003e - Cardiac rough ER is a critical subdomain of ER, and in cardiomyocytes has a predominantly perinuclear morphology that is distinct from the repeating SR sarcomeres that control contraction [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Yet, SERCA-positive SR membrane vesicles also contained a complete collection of known rough ER proteins involved in translation, translocation, and N-linked glycosylation (Table\u0026nbsp;6, \u003cem\u003eAppendix\u003c/em\u003e). Rough ER proteins were of relatively low abundance (A\u003csub\u003eSR\u003c/sub\u003e= 4.0 \u0026plusmn; 1.7), but very highly-enriched over crude MVs (E\u003csub\u003eSR\u003c/sub\u003e= 6.0 \u0026plusmn; 2.5) (Fig.\u0026nbsp;8A).\u003c/p\u003e \u003cp\u003e \u003cem\u003e5.6 Lipid metabolism and lipid modifications of proteins -\u003c/em\u003e Numerous enzymes involved in lipid metabolism were enriched in SERCA-positive membranes. The highest A\u003csub\u003eSR\u003c/sub\u003e value (=\u0026thinsp;20.7) resulted from CDITP, which appends inositol-3-phosphate to diacylglycerol, with numerous lipid metabolizing proteins present at lower A\u003csub\u003eSR\u003c/sub\u003e levels (Fig.\u0026nbsp;9, \u003cem\u003eAppendix\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e \u003cem\u003e5.7 Proteins involved in ER membrane structure and dynamics -\u003c/em\u003e Many cardiac ER/SR proteins are those thought to play roles in distributing and trafficking proteins across the biosynthetic pathway. Several act by guiding membrane patches along transport filaments; these include Ca-binding protein p22, vesicle-trafficking protein Sec22b, vesicle-associated membrane protein 2 (VAMP-2), and vesicle transport protein Sec20 [\u003cspan additionalcitationids=\"CR68 CR69\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The mammalian proteins of the p24 family (TMED 10, 9, 2, 1) are involved in selective loading of cargo in transport vesicle between membrane compartments, and the co-enrichment and relative abundances of its known subunits support a role in cardiac ER/SR protein distribution [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Finally, other ER proteins may function in the maintaining the structure of ER subcompartments, such as reticulons-2 and \u0026minus;\u0026thinsp;4 [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], lunapark-3 [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], and climp-63 [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Proteins involved in ER/SR dynamics are tabulated, along with enrichment values, in Table\u0026nbsp;7, \u003cem\u003eAppendix\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Conclusions","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cp\u003eIn this study, we used the technique of Ca oxalate loading of cardiac SR membrane vesicles to produce two SR membrane subpopulations (MedSR and HighSR), thereby enriching patches of cardiac ER/SR membrane that contain combinations of SERCA \u003cem\u003eand\u003c/em\u003e RyR levels. Roughly one third of microsomal proteins were enriched in SERCA-positive membranes, and about a third of those were more enriched in the MedSR membranes, indicating the presence of enriched RyR in the same SR patch, and suggesting a relative proximity to junctional SR, or at least a biochemically distinct membrane subdomain. The activation of SERCA activity in our current study, an historical measure of SR function, led to the enrichment of proteins from every ER subcompartment, supporting a view that cardiac ER and SR should cannot be demarcated based only upon their ability to function in Ca handling.\u003c/p\u003e \u003cp\u003eProteins enriched in SERCA-positive SR vesicles were defined as any protein that was enriched \u0026ge;2.0-fold over its level in crude heart microsomes. This single enrichment criterion selected 354 proteins of 1102 total proteins in crude microsomes, while excluding all mitochondrial, contractile protein, and other known organellar contaminants. Even major mitochondrial and contractile protein contaminants were eliminated by this simple measure of enrichment. SERCA-positive SR proteins encompassed proteins from all known functional ER and SR subdomains, leading us to conclude that SERCA-positive membranes represent cardiac ER and SR. Plotting A\u003csub\u003eSR\u003c/sub\u003e values for the top 2\u0026ndash;4 proteins of different groups of proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e), provided some semi-quantitative insight into how different ER/SR subdomains may contribute to overall SR function.\u003c/p\u003e \u003cp\u003eIn spite of these substantial variations in A\u003csub\u003eSR\u003c/sub\u003e among functional sets of proteins, the enrichment properties E\u003csub\u003esub\u003c/sub\u003e and E\u003csub\u003eSR\u003c/sub\u003e among the same sets were remarkably consistent; both because of their physical segregation in membrane vesicles, but also because enrichment values are derived from \u003cem\u003eratios\u003c/em\u003e of A\u003csub\u003eSR\u003c/sub\u003e values in two preparations. For example, a large number of known KDEL proteins were found in SR, exhibiting a wide range of A\u003csub\u003eSR\u003c/sub\u003e values (6.3 \u0026plusmn;9.0, N\u0026thinsp;=\u0026thinsp;7 proteins). For the same 7 KDEL proteins, however, E\u003csub\u003esub\u003c/sub\u003e values were 0.47 \u0026plusmn;0.08, and E\u003csub\u003eSR\u003c/sub\u003e values were 7.1 \u0026plusmn;3.7.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe enrichment of junctional SR proteins in MedSR membranes was previously demonstrated by immunoblot analyses to be a feature of this SR preparation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. In the present study, we found that the values of E\u003csub\u003esub\u003c/sub\u003e for 354 proteins formed a continuous range from \u0026minus;\u0026thinsp;0.9 (largely detected only in MedSR) to +\u0026thinsp;0.9 (largely detected only in HighSR membranes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Other functional protein groups exhibited similarly segregated values (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,8), suggesting that they too were physically distributed into at least two divergent subcellular sites: those closer to junctional SR and those further removed from such sites. The segregation of protein functional groups in terms of E\u003csub\u003eSR\u003c/sub\u003e and E\u003csub\u003esub\u003c/sub\u003e shows that vesicles are derived from small enough membrane surfaces to fractionate with different enrichment patterns, and do not simply represent huge sections of membrane surface.\u003c/p\u003e \u003cp\u003eIn summary, enrichment of cardiac membranes by Ca oxalate loading leads to the enrichment of ER/SR proteins, distributed between membrane fractions that differ in Ca leak through RyR. The distribution of individual proteins between the two fractions (Esub), and the consistent enrichments found among different functional sets of proteins, reflects the connections between ER/SR subdomains and the well-studied spatial relationships between junctional and free SR. Our data present for the first time a reliable estimation of cardiac ER/SR protein content, along with a semi-quantitative assessment of prominent sets of functional ER/SR subdomains present in a microsomal preparation of canine ventricular tissue.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the support of a grant from the Office of the Vice President for Research at Wayne State University (S.C.). Assistance of the Wayne State University Proteomics Core was supported through NIH grants P30 ES020957, P30 CA 022453 and S10 OD010700. This work was also supported by the National Institutes of Health [1R01DK110314 (X. C.)], and the American Heart Association. [18TPA34170284 /ZC/2018].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSteven Cala:\u003c/em\u003e Conceptualization, Methodology, Writing. \u003cem\u003eNicholas Carruthers:\u003c/em\u003e Data curation, Software\u003cem\u003e. \u0026nbsp;Paul Stemmer:\u003c/em\u003e Supervision, Investigation, Methodology. \u003cem\u003eZhenhui Chen:\u003c/em\u003e Validation, Investigation. \u003cem\u003eXuequn Chen:\u0026nbsp;\u003c/em\u003eResources, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available\u0026nbsp;at Figshare.com; Filename: CardiacSRproteome; DOI: 10.6084/m9.figshare.19953701. Raw data and search results have been submitted to the ProteomeXchange via PRIDE with accession PXD022455 (Username: [email protected], Password: G1wlk66N). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animal research was approved by the Wayne State University Animal Investigation Committee (protocol #A 04-02-13).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBennett HS and Porter KR (1953) An electron microscope study of sectioned breast muscle of the domestic fowl. Am J Anat 93:61-105. doi: 10.1002/aja.1000930104\u003c/li\u003e\n\u003cli\u003ePorter KR and Palade GE (1957) Studies on the endoplasmic reticulum. III. Its form and distribution in striated muscle cells. 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J Biol Chem 272:23389-97. doi: 10.1074/jbc.272.37.23389\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Materials","content":"\u003cp\u003eOnline Resource 1 is not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cardiac sarcoplasmic reticulum, endoplasmic, SERCA, ryanodine receptor, proteome","lastPublishedDoi":"10.21203/rs.3.rs-2557992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2557992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe importance of sarcoplasmic reticulum (SR) Ca-handling in heart has led to detailed understanding of Ca-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Analyses of protein enrichments from the 3 membrane preparations (crude microsomes, MedSR, and HighSR), showed that only a third of microsomal proteins in heart, or 354 proteins, were enriched \u0026ge;2.0-fold in SR. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca leak (ryanodine receptor) co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins from other putative ER/SR subdomains also showed characteristic distributions among SR subpopulations. We conclude that active Ca loading of cardiac microsomes, reflecting the combined activities of Ca uptake by SERCA, and Ca leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.\u003c/p\u003e","manuscriptTitle":"Activation of Ca transport in cardiac microsomes enriches functional sets of ER and SR proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-08 18:06:24","doi":"10.21203/rs.3.rs-2557992/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-02-20T00:23:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-02-14T17:51:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44f685c4-69fc-4dbe-9e71-67fec4bd6952","date":"2023-02-07T23:12:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-02-07T22:01:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-07T21:52:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-02-07T06:44:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2023-02-07T01:50:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bc1cfebe-1e07-4e1a-a396-2ded93af9c11","owner":[],"postedDate":"February 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:30:38+00:00","versionOfRecord":{"articleIdentity":"rs-2557992","link":"https://doi.org/10.1007/s11010-023-04708-0","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2023-04-10 20:26:23","publishedOnDateReadable":"April 10th, 2023"},"versionCreatedAt":"2023-02-08 18:06:24","video":"","vorDoi":"10.1007/s11010-023-04708-0","vorDoiUrl":"https://doi.org/10.1007/s11010-023-04708-0","workflowStages":[]},"version":"v1","identity":"rs-2557992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2557992","identity":"rs-2557992","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}