©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification, Primary Structure, and Immunological Characterization of the 26-kDa Calsequestrin Binding Protein (Junctin) from Cardiac Junctional Sarcoplasmic Reticulum (*)

(Received for publication, August 30, 1995)

Larry R. Jones (1)(§) Lin Zhang (1) Kristi Sanborn (1) Annelise O. Jorgensen (2) Jeff Kelley (1)

From the  (1)Department of Medicine and the Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the (2)Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario M5S IA8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previously we identified a protein of apparent M(r) = 26,000 as the major calsequestrin binding protein in junctional sarcoplasmic reticulum vesicles isolated from cardiac and skeletal muscle (Mitchell, R. D., Simmerman, H. K. B., and Jones, L. R.(1988) J. Biol. Chem. 263, 1376-1381). Here we describe the purification and primary structure of the 26-kDa calsequestrin binding protein. The protein was purified 164-fold from cardiac microsomes and shown by immunoblotting to be highly enriched in junctional membrane subfractions. It ran as a closely spaced doublet on SDS-polyacrylamide gel electrophoresis and bound I-calsequestrin intensely. Cloning of the cDNA predicted a protein of 210 amino acids containing a single transmembrane domain. The protein has a short N-terminal region located in the cytoplasm, and the bulk of the molecule, which is highly charged and basic, projects into the sarcoplasmic reticulum lumen. Significant homologies were found with triadin and aspartyl beta-hydroxylase, suggesting that all three proteins are members of a family of single membrane-spanning endoplasmic reticulum proteins. Immunocytochemical labeling localized the 26-kDa protein to junctional sarcoplasmic reticulum in cardiac and skeletal muscle. The same gene product was expressed in these two tissues. The calsequestrin binding activity of the 26-kDa protein combined with its codistribution with calsequestrin and ryanodine receptors strongly suggests that the protein plays an important role in the organization and/or function of the Ca release complex. Because the 26-kDa calsequestrin binding protein is an integral component of the junctional sarcoplasmic reticulum membrane in cardiac and skeletal muscle, we have named it Junctin.


INTRODUCTION

The technique of membrane subcellular fractionation has proven to be invaluable for biochemical dissection of the molecular components of the SR (^1)responsible for Ca uptake and Ca release in striated muscle(1, 2) . Utilizing this approach it is possible to isolate biochemically subspecialized regions of SR in the form of sealed membrane vesicles from both cardiac (3) and skeletal muscle(4) . Free SR vesicles originate from the region of the SR surrounding the myofibrils. Ca is actively transported here into the SR lumen(5, 6) . Junctional SR vesicles arise from the regions of the SR making contact with the sarcolemma where Ca is released to initiate muscle contraction (1, 2, 6, 7) . A few of the junctional SR proteins participating in Ca release have been purified and cloned(1, 2) . Ryanodine receptors, large tetrameric proteins of subunit molecular weight approximately one-half million, are the Ca release channels. Ryanodine receptors constitute the SR feet, structures that join the junctional SR membrane to transverse tubules and regions of surface sarcolemma in intact muscle. CSQ, a peripheral membrane protein, is a high capacity Ca binding protein located in the lumen of the junctional SR(1, 2) . CSQ is visible as an electron-dense matrix, which appears to be associated with the inner surface of the junctional SR membrane(1, 6) . CSQ acts as a Ca reservoir, providing a ready source for Ca when ryanodine receptors open and Ca is released into the cytoplasm. Although both ryanodine receptors (9, 10) and CSQ(11, 12) are the products of separate genes in cardiac and skeletal muscle, the molecular architecture at the junctional region in each tissue is similar(1, 6) . The close proximity of CSQ to ryanodine receptors in striated muscle suggests that specific protein interactions are required for stabilization and efficient operation of the Ca release process. The membrane surface where these protein interactions occur is called the junctional face membrane (8) .

Identification of all of the molecular components of the junctional face membrane important for Ca release is an area of intense study(1, 2, 7) . ``Rope-like fibers'' (8) or ``joining strands'' (13) have been visualized by electron microscopy, which appear to anchor CSQ to the junctional membrane at sites apposed to ryanodine receptors. Recently, it has been proposed that triadin, a 95-kDa junctional SR glycoprotein(14, 15, 16, 17) , may be a component of these anchoring strands by virtue of its ability to bind both CSQ and ryanodine receptors(18) . In an earlier study, we described a different CSQ binding protein localized to both canine cardiac and rabbit fast skeletal muscle junctional SR vesicles(19) . This protein was detected by binding I-labeled CSQ to junctional SR proteins transferred to nitrocellulose. It was the major CSQ binding protein identified and migrated as a closely spaced doublet of apparent molecular weight 26,000. Subsequently, using the same approach, Damiani and Margreth (20) confirmed the presence of the 26-kDa CSQ binding protein in junctional face membranes prepared from rabbit fast skeletal muscle. A similar 26-kDa protein doublet had been described earlier as a major constituent of this membrane(8) . A protein doublet of approximately the same molecular weight, which appeared to interact functionally with CSQ and regulate the amount of Ca released from junctional SR vesicles by caffeine, was also recently reported by Ikemoto et al.(21) .

Here we have purified the 26-kDa CSQ binding protein and deduced its complete primary structure by cDNA cloning. We show that the same protein is expressed in heart and skeletal muscle and with use of specific antibodies localize the protein to the junctional SR in these two tissues. This newly characterized protein of the junctional face membrane shares many similarities with triadin and is likely to play an important role in Ca release in striated muscle. A preliminary report on purification of the 26-kDa CSQ binding protein appeared in abstract form(23) .


EXPERIMENTAL PROCEDURES

Isolation of Membrane Subfractions

Procedure I microsomes were isolated from canine left ventricle(22) . Subfractionation of these microsomes was by Ca oxalate loading followed by sucrose density gradient centrifugation(3, 22) . Subfractions A, B, C, and D were collected at the 0.6, 0.8, 1.0, and 1.5 M sucrose interfaces, respectively. Subfraction E was obtained as the pellet below 1.5 M sucrose. Subfractions A, D, and E are enriched in sarcolemmal vesicles, and junctional and free SR vesicles, respectively(3, 22, 24) . Further purification of junctional SR vesicles (subfraction D membranes) was achieved by a second round of Ca oxalate loading, this time in the presence of 300 µM ryanodine, to effect a ryanodine-mediated density shift(25) . Following Ca oxalate loading of subfraction D microsomes in the presence of ryanodine, the membranes were centrifuged through a 1.3 M sucrose cushion. P, T, and B designate the starting parent (P) membranes (or subfraction D microsomes), the membranes collected on top of the 1.3 M sucrose cushion (T), and the membranes pelleted at the bottom (B) of the sucrose layer. As a control, subfraction D microsomes were also loaded in the absence of ryanodine. Procedure I microsomes were also prepared from canine fast twitch (tensor fasciae latae) and slow twitch (vastus intermedius) skeletal muscle. Canine liver microsomes were obtained as the membranes sedimenting between 10,000 and 100,000 times g(max)(25) . All membranes were stored frozen at -40 °C in small aliquots in 0.25 M sucrose, 30 mM histidine (pH 7.2).

Solubilization of the 26-kDa CSQ Binding Protein

32 µl (166 µg of protein) of subfraction D membranes in 0.25 M sucrose, 30 mM histidine (pH 7.2) were added to a Beckman TL-100 centrifuge tube, followed by addition of 8 µl of 10% Triton X-100. 13 µl of sample was saved for analysis, and the remainder was centrifuged at 100,000 rpm for 5 min. The supernatant (S(1)) was collected, and the pellet (P(1)) was resuspended in 27 µl of 20 mM MOPS, 0.5 M NaCl, and 2% Triton X-100. 13 µl was saved, and the remainder was spun at 100,000 rpm for 10 min to yield supernatant (S(2)) and pellet (P(2)) fractions. P(2) was resuspended in 13 µl of 0.25 M sucrose. 13 µl from all fractions were analyzed for I-CSQ binding as described below.

Purification of the 26-kDa CSQ Binding Protein

For a typical purification, 214 mg of procedure I canine cardiac microsomal protein at a concentration of 7.74 mg/ml was solubilized at room temperature for 10 min in 0.22 M sucrose, 26 mM histidine, 0.5 M NaCl, and 2% Triton X-100. The sample was sedimented at 45,000 rpm for 30 min in a Beckman Ti 70 rotor. The supernatant was diluted with an equal volume (27.7 ml) of 20 mM MOPS, 0.1% Triton X-100 (pH 7.2) (Buffer A) and loaded over a 20-ml phosphocellulose column preequilibrated with Buffer A. The flow-through fraction was collected, and the column was eluted with 20-ml washes of Buffer A containing 0.2-0.9 M NaCl applied in 0.1 M concentration steps. Aliquots of the column fractions were electrophoresed and transferred to nitrocellulose to localize I-CSQ binding proteins, as described below. The 0.5-0.8 M NaCl wash fractions containing the partially purified 26-kDa CSQ binding protein were pooled and concentrated, and the sample was subjected to preparative SDS-PAGE using a Bio-Rad 491 Prep Cell(25) . The PAGE fractions containing the purified 26-kDa protein were identified by I-CSQ binding assay and combined. In the typical preparation described 150 µg of purified 26-kDa CSQ binding protein was isolated. Protein concentration was determined by the Amido Black method(27) .

SDS-PAGE and I-CSQ Binding

SDS-PAGE was conducted with a 7% polyacrylamide gel according to Porzio and Pearson (28) ( Fig. 1only) or by the method of Laemmli (26) with 9 or 10% separating gels and a 3% stacking gel. Gels were stained with Coomassie Blue or, for detection of I-CSQ binding proteins, proteins were transferred to nitrocellulose(19) . Nitrocellulose sheets were stained with Amido Black (29) to localize proteins and then blocked two times for 30 min in 20 mM MOPS, 150 mM KCl (pH 7.2) (Buffer B) containing 1% horse hemoglobin. Blots were washed three times for 5 min in 30 ml of Buffer B containing 1 mM EGTA, and then 5-10 µg of I-CSQ in 50 µl (approximately 8 times 10^6 cpm) were added, and the blot was incubated for 90 min at room temperature. After incubation with radioactive CSQ, blots were washed three times for 15 min in Buffer B containing 1 mM EGTA, and then autoradiography was performed. Canine cardiac CSQ was prepared by phenyl-Sepharose chromatography (30) and iodinated using Enzymobeads(19) .


Figure 1: Solubilization of the 26-kDa CSQ binding protein. An autoradiograph of I-CSQ binding to canine cardiac junctional SR proteins transferred to nitrocellulose is shown. Subfraction D (JSR) was extracted with 2% Triton X-100 to yield supernatant (S(1)) and pellet (P(1)) fractions. P(1) was extracted with 2% Triton X-100 plus 0.5 M NaCl to yield secondary supernatant (S(2)) and pellet (S(2)) fractions. Equivalent volumes of fractions were loaded on the gel. Large arrow indicates the 26-kDa CSQ binding protein, and the small arrowheads designate cardiac isoforms of triadin, substantiated by binding of a triadin antibody (GP-58K) to both of these bands and by the partial amino acid sequence obtained from the higher mobility isoform.^2 The asterisk denotes a minor CSQ binding band, which appears to be related to the 26-kDa CSQ binding protein.



Protein and Peptide Sequencing

50-100 µg of purified 26-kDa CSQ binding protein was precipitated (31) and resuspended in 60% n-propyl alcohol, 0.1% trifluoroacetic acid for direct sequence analysis or in 200 µl of 20 mM Tris, 0.1% hydrogenated Triton X-100 (pH 8.5) (32) for digestion with proteases. Trypsin, endoproteinase Lys-C, or endoproteinase Asp-N was added at 1/20 weight ratios, and digestions were conducted overnight at 37 °C. Peptides were separated by C(18) reverse-phase chromatography using a Pharmacia Biotech Inc. PepRPC column and sequenced by Edman degradation with an Applied Biosystems model 477A automated sequencer(25) .

Antibody Production and Immunoblotting

Antibodies were produced to the purified 26-kDa CSQ binding protein and to synthetic peptides made to residues 77-91 (Peptide 1) (EGPGGVAKRKTKAKV) and to residues 94-108 (Peptide 2) (LTKEELKKEKEKTES). Peptides were synthesized with N-terminal cysteine residues and coupled to thyroglobulin and bovine serum albumin(33) . Affinity purification of antibodies was by the method of Olmsted(34) . Immunoblotting was done as described previously(35, 36) . Monoclonal antibody 2A7-A1 was produced to the Ca-ATPase in canine cardiac SR vesicles(37) .

Immunofluorescence Labeling

Immunofluorescence labeling with affinity-purified rabbit antibodies to the intact 26-kDa CSQ binding protein was performed as described previously(38, 39) . To localize the fast (type II) isoform of the SR Ca-ATPase, mouse monoclonal antibody IIH11 was used(40) . Confocal microscopy was carried out as recently described. (^2)

Trypsin Digestion of Junctional SR Vesicles

10 µg of procedure I microsomes from dog heart were incubated at room temperature for 30 min in 50 mM MOPS (pH 7.0), 3 mM MgCl(2), 100 mM KCl, 0.1 mM CaCl(2), and 0.5 µg of trypsin in the presence and absence of 0.2% Triton X-100. Control samples were incubated without trypsin and Triton X-100. Reactions were quenched by boiling in Laemmli solubilization buffer containing 10% SDS. Samples were analyzed by immunoblotting.

cDNA Cloning and Sequencing

A canine cardiac gt11 expression library was screened with affinity-purified antisera against the 26-kDa CSQ binding protein and peptide 1 as described previously (11) . Positive phage clones were plaque-purified, and DNA was prepared by the plate lysate method(41) . EcoRI inserts were subcloned into pBluescript. Clones from a canine skeletal muscle ZAPII cDNA library (Stratagene) were identified with affinity-purified antibodies to the intact protein. A canine cardiac gt10 cDNA library (42) was screened with a P-labeled synthetic oligonucleotide probe corresponding to the 5`-end of clone 40 (bp -80 to -51 (see Fig. 8)). Double-stranded sequencing of all clones was performed by the dideoxy method(43) , using universal plasmid primers and a series of internal synthetic primers(41) . Sequence analyses and data base searches were conducted with software from MacVector.


Figure 8: Nucleotide and deduced amino acid sequence of the 26-kDa CSQ binding protein. The start (ATG) and stop (TGA) codons and the first methionine residue (M) are in boldface. Polyadenylation signals are boldface and underlined. Underlined amino acids were confirmed by N-terminal sequencing the intact protein or proteolytic peptides purified by reverse-phase chromatography. Dots denote four amino acid residues not confirmed. The double underline marks the predicted transmembrane domain.



Northern Blot Analysis

30 µg of total RNA prepared from canine heart and skeletal muscle were electrophoresed in a 0.8% agarose gel containing 2.2 M formaldehyde and transferred to a GeneScreen membrane. Hybridization was with a P-labeled probe made to the EcoRI insert in clone 8 (see Fig. 7). Autoradiography was performed at -80 °C for 3 days with an intensifying screen.


Figure 7: Restriction map of 26-kDa CSQ binding protein cDNA. Solid black bars denote open reading frames with start (ATG) and stop (TGA) codons indicated for full-length Clone 2. The striped boxes and A indicate polyadenylation signals and tails, respectively. All clones were isolated from canine libraries, either cardiac or skeletal muscle, as indicated.



[^3H]Ryanodine Binding

[^3H]Ryanodine binding to 40 µg of membrane subfractions was performed by filtration assay in 0.6 M NaCl, 1 mM CaCl(2), 20 mM MOPS, and 20 nM radioactive ligand (pH 7.1)(44) . Nonspecific binding was determined in the same medium containing 10 µM non-radioactive ryanodine.


RESULTS

Solubilization and Purification of the 26-kDa CSQ Binding Protein from Canine Cardiac SR

Consistent with our earlier study(19) , a major radioactive protein band of apparent M(r) = 26,000 was observed when canine cardiac junctional SR vesicles were transferred to nitrocellulose and incubated with I-CSQ in Ca-free medium (Fig. 1, arrow). Several other minor I-labeled bands were also detected. The two radioactive bands running just below the 45-kDa protein standard (arrowheads) are cardiac isoforms of triadin.^2 (These two I-CSQ binding proteins were designated bands 4 and 5 in our earlier study ( Fig. 11of (19) ).) Another minor labeled band was detected migrating just above the 31-kDa protein standard (asterisk). This minor band may represent a protein isoform of the 26-kDa CSQ binding protein but is not considered further in this work. 2% Triton X-100 was insufficient to solubilize a significant amount of the 26-kDa CSQ binding protein from junctional SR vesicles (Fig. 1, S(1)). With addition of 0.5 M NaCl, however, most of the protein became soluble (Fig. 1, S(2)). The requirement of a relatively high concentration of detergent plus a high ionic strength medium for efficient solubilization of the 26-kDa CSQ binding protein suggested that it was an integral membrane protein. Consistent with this, 100 mM sodium carbonate at pH 11.4 also failed to solubilize the protein.


Figure 11: Sequence alignment of aspartyl beta-hydroxylase (OHase), Junctin (Junct), and triadin (Triad). Identical residues in all three proteins are shaded. Different residues are marked by vertical ticks. Dashes in the Junctin sequence denote a gap for proper alignment with OHase. Residue numbers for the three proteins are given at the left and right margins. For triadin, only residues spanning the region of homology are shown.



For purification of the 26-kDa CSQ binding protein, we used procedure I cardiac membrane vesicles as the starting material, which is a heterogeneous microsomal preparation consisting mostly of sarcolemmal and free and junctional SR vesicles(3, 22, 24) . The detergent extract from these membranes was loaded over a phosphocellulose column (Fig. 2). All of the 26-kDa CSQ binding protein was retained by the column and was subsequently eluted in the 0.5-0.7 M NaCl wash fractions. A closely spaced doublet corresponding to the 26-kDa CSQ binding protein was visible in these fractions by Coomassie Blue staining (Fig. 2A) and also by I-CSQ binding (Fig. 2B). (Both bands of the 26-kDa doublet reacted equally with labeled CSQ but are not resolved in the photograph.) The proteins corresponding to the cardiac isoforms of triadin (arrowheads) eluted from the column just before the 26-kDa CSQ binding protein in the 0.4 and 0.5 M NaCl wash fractions (Fig. 2B). The 31-kDa CSQ binding protein eluted in the same fraction as the 26-kDa protein (asterisk). Of several different types of chromatography tried for purification of the 26-kDa protein, only phosphocellulose gave a substantial enrichment. The solubilized protein did not bind to concanavalin A- or wheat germ agglutinin-agarose.


Figure 2: Purification of the 26-kDa CSQ binding protein. A shows a Coomassie Blue-stained polyacrylamide gel containing procedure I cardiac membrane vesicles (MV), and pellet (P) and supernatant (S) fractions obtained after detergent extraction (Extract) of these membranes. Cellulose Phosphate indicates fractions eluting from the phosphocellulose column, including the flow-through (Ft) fraction and 0.2-0.9M NaCl wash fractions. Equivalent volumes of all fractions were loaded on the gel. SDS designates the purified 26-kDa protein doublet, obtained after preparative gel electrophoresis. B is an autoradiograph of identical samples transferred to nitrocellulose and incubated with I-CSQ. Large arrow indicates the 26-kDa protein doublet. Arrowheads designate cardiac isoforms of triadin. The asterisk denotes the CSQ binding protein of approximately 31 kDa, which co-purified on phosphocellulose with the 26-kDa protein.



After pooling and concentrating the 0.5-0.7 M NaCl wash fractions from the phosphocellulose column, the 26-kDa CSQ binding protein was purified to homogeneity in one additional step by preparative SDS-PAGE. The purified protein reacted strongly with I-CSQ and continued to run as a closely spaced doublet (Fig. 2, SDS). Quantification of I-CSQ binding to the protein revealed that it was purified 164-fold from procedure I membranes. 135 ± 18 µg (mean ± S.E. from 10 preparations) of purified protein were routinely isolated from 200 mg of cardiac microsomes.

Immunoblot Analysis of 26-kDa CSQ Binding Protein in Different Membrane Subfractions

Rabbit antiserum was produced to the purified 26-kDa CSQ binding protein, which recognized the expected 26-kDa protein doublet in procedure I cardiac membranes (Fig. 3A). Relative to procedure I membranes, antibody binding to the 26-kDa protein was increased 7.3-fold in subfraction D, which is enriched in junctional SR vesicles(3, 24) . Subfraction E, which contains mostly free SR vesicles(3, 24) , contained only 19% of the binding activity of subfraction D. [^3H]Ryanodine binding to subfraction D (10.2 pmol/mg of protein) was increased 4.0-fold over that in procedure I membranes; subfraction E contained only 16% of the [^3H]ryanodine binding activity of subfraction D (see the legend to Fig. 3). The Ca pump was approximately equally distributed between subfraction D and subfraction E (Fig. 3B). These results demonstrated that the 26-kDa CSQ binding protein and ryanodine receptors co-purified in the junctional SR subfraction isolated from canine heart. The co-localization of CSQ to the same junctional SR subfraction was shown previously(3, 24) . The 164-fold purification of the 26-kDa CSQ binding protein from crude SR vesicles, coupled with the 7.3-fold enrichment of the protein in subfraction D, suggests that the 26-kDa CSQ binding protein contributes 4.4% of the total protein of junctional SR vesicles.


Figure 3: Immunoblots of 26-kDa CSQ binding protein in cardiac membrane subfractions. A is an immunoblot showing the 26-kDa protein doublet in procedure I cardiac membrane vesicles (MV) and in subfractions A-E isolated by Ca oxalate loading followed by density gradient centrifugation. 10 µg of all fractions were loaded on the gel. Affinity-purified antibodies to the intact 26-kDa protein were used. Band intensities were quantified with use of a GS-250 molecular imager (Bio-Rad). Specific [^3H]ryanodine binding values (pmol/mg protein) of the same fractions were: MV, 2.55; A, 2.08; B, 1.64; C, 3.48; D, 10.2; and E, 1.61. B shows results after the same blot was erased and probed with monoclonal antibody 2A7-A1 to the Ca-ATPase. Only the region of the nitrocellulose membrane binding the antibody is shown. C is an immunoblot showing recovery of the 26-kDa CSQ binding protein doublet in parent (P), top (T), and bottom (B) fractions, after subfraction D membranes were loaded with Ca oxalate in the presence (+RY) and absence (-RY) of 300 µM ryanodine and centrifuged through 1.3 M sucrose. Affinity-purified antibodies to the intact protein were used. Equivalent volumes of all fractions were loaded so that autoradiographic intensities reflect 26-kDa protein recoveries.



To obtain further evidence that the 26-kDa CSQ binding protein was preferentially localized to junctional SR vesicles in heart, we subjected the D subfraction of membranes already enriched in this protein to Ca oxalate loading in the presence of ryanodine to effect a density shift(25) . When actively transporting Ca, cardiac junctional SR vesicles are unable to accumulate significant amounts of Ca oxalate in the absence of ryanodine because Ca transported inside the vesicles effluxes through open Ca release channels (ryanodine receptors). When 300 µM ryanodine is added to the uptake buffer, Ca release channels are blocked and Ca oxalate is accumulated(3, 24) , increasing the density of the vesicles(25) . When subfraction D membranes were loaded with Ca oxalate in the absence of ryanodine, most of the membrane protein including the 26-kDa CSQ binding protein was recovered on top of the 1.3 M sucrose cushion (Fig. 3C). When vesicles were loaded in the presence of ryanodine, virtually all of the 26-kDa CSQ binding protein pelleted through the 1.3 M sucrose cushion along with 36% of the starting membrane protein material. These results confirm that the 26-kDa CSQ binding protein is localized to those SR vesicles sensitive to ryanodine.

To determine if the 26-kDa protein was present in skeletal muscle SR vesicles, we performed immunoblots with dog cardiac, fast skeletal, and slow skeletal muscle microsomes. Liver microsomes were also analyzed. Antibodies to the intact protein were used, as well as to peptide 1 (residues 77-91) and peptide 2 (residues 94-108). The same 26-kDa protein doublet was detected in all three striated muscle fractions with use of antibody to the whole protein (Fig. 4A), to peptide 1 (Fig. 4B), and to peptide 2 (Fig. 4C). Fast skeletal muscle membranes were most enriched in the protein, followed by slow skeletal muscle membranes and then cardiac membranes. These results strongly suggest that the same 26-kDa CSQ binding protein is present in both cardiac and skeletal muscle SR membranes. The 26-kDa protein was not detected in canine liver microsomes (Fig. 4). Using the antiserum generated to the whole protein and to peptide 1, we also detected 26-kDa antibody binding bands in membranes from dog atrium, rabbit ventricle and skeletal muscle, and guinea pig, rat, and mouse ventricles (data not shown).


Figure 4: Tissue distribution of 26-kDa CSQ binding protein. 40 µg of microsomal protein from dog heart (C), slow skeletal muscle (S), fast skeletal muscle (F), and liver (L) were electrophoresed, transferred to nitrocellulose, and incubated with affinity-purified antibodies to the intact 26-kDa CSQ binding protein (A), peptide 1 (B), and peptide 2 (C). Arrowhead points to the 26-kDa protein doublet. Cross-reacting bands of higher molecular weight visible in C are nonspecific and varied in intensity from experiment to experiment.



Immunofluorescence Localization of the 26-kDa CSQ Binding Protein

The cellular and subcellular distribution of the 26-kDa CSQ binding protein was probed by indirect immunofluorescence labeling of cryosections of canine ventricular and skeletal muscle tissue using affinity-purified antibodies to the intact protein.

Confocal imaging of cardiac papillary muscle showed that specific labeling was densely distributed in canine ventricular myofibers (Fig. 5, a-d). The intensity of labeling of vascular smooth muscle cells (Fig. 5a, sm), endothelial cells (Fig. 5a, E), and fibroblasts (not shown) was indistinguishable from that of the background (not shown). Confocal imaging of longitudinal cryosections showed that specific labeling was mainly confined to rows of discrete foci oriented transversely to the longitudinal axis of the myofibers (Fig. 5, c and d). Phase contrast microscopical imaging of the same field showed that most of the discrete immunofluorescently labeled foci (Fig. 5d, white arrows) were localized to the center of the I band region (Fig. 5e, white arrows), where most of the junctional and corbular SR (6, 45, 46) are localized in mammalian cardiac myofibers. In transversely cut ventricular myofibers specific labeling of the 26-kDa CSQ binding protein was confined to discrete foci distributed in a network-like pattern throughout the cytoplasm (Fig. 5b). The finding that the subcellular distribution of the 26-kDa CSQ binding protein in ventricular myocytes corresponds very closely to that previously reported for CSQ (45, 47) and the ryanodine receptor (46) suggests that this protein is localized to junctional and/or corbular SR in cardiac myocytes.


Figure 5: Immunofluorescence labeling of the 26-kDa CSQ binding protein in canine ventricular papillary muscle. Transverse (a and b) and longitudinal (c-e) cryosections of canine ventricular muscle were labeled by immunofluorescence with affinity-purified antibodies to the intact 26-kDa CSQ binding protein and imaged by confocal microscopy (a-d) and phase contrast microscopy (e). Specific labeling was confined to ventricular myofibers (M) (a-c) while labeling of endothelial cells (E) and arterial smooth muscle cells (sm) was indistinguishable from background (a). Comparison of the staining pattern of longitudinal sections of papillary myofibers (d) with the position of the A and I bands in the same field imaged by phase contrast microscopy (e) showed that discrete fluorescent foci were localized at the center of the I band region (white arrows) where junctional SR and corbular SR are densely distributed in mammalian cardiac myofibers. Magnifications: a and c, times 600; b, times 1,100; d and e, times 1,650. Scale bar, 10 µm.



Immunofluorescence labeling of serial transverse sections of canine gracilis muscle containing both fast and slow skeletal muscle fibers showed that the 26-kDa CSQ binding protein was densely distributed in all myofibers (Fig. 6a) but not detected in smooth muscle cells, endothelial cells, and fibroblasts present in the same tissue section (not shown). The intensity of labeling was slightly higher in fast fibers (black dots) than in slow fibers (white dots). These results were in agreement with the immunoblot analysis showing that the 26-kDa CSQ binding protein was present in SR vesicles from fast and slow skeletal muscle (Fig. 4). Examination of transverse sections of fast skeletal myofibers at higher magnification showed a polygonal fluorescent staining pattern throughout the cytoplasm of the myofibers (Fig. 6c). The center to center distance between neighboring polygons ranged from 1.1 to 1.5 µm, which is close to the center to center distance between neighboring myofibrils in adult skeletal muscle. In longitudinal cryosections of canine gracilis muscle immunofluorescence labeling appeared as transversely oriented rows of discrete foci (Fig. 6, d and f). The position of the transversely oriented rows of fluorescent foci (Fig. 6d, white arrows) corresponded to the interface between A bands and I bands as determined by imaging the same field by phase contrast microscopy (Fig. 6e). In contrast the intensity of labeling of the cell surface (Fig. 6d, white arrowheads) was indistinguishable from that of the background (not shown). Thus the distribution of immunofluorescence labeling of the 26-kDa CSQ binding protein in skeletal muscle also corresponded very closely to that of CSQ (39, 48) and the ryanodine receptor(7, 49, 50) .


Figure 6: Immunofluorescence labeling of adult canine skeletal (gracilis) muscle. Transverse (a-c) and longitudinal (d-f) cryosections of canine gracilis muscle were immunofluorescently stained with affinity-purified antibodies to the 26-kDa CSQ binding protein (a, c, d, and f) or monoclonal antibody IIH11 to the fast isoform of the Ca-ATPase (b). The sections were imaged by confocal (a-d and f) and phase contrast (e) microscopy. Black dots and white dots denote fast and slow muscle fibers, respectively (a and b). Specific labeling for the 26-kDa protein in longitudinal sections (d and f) was mainly confined to transversely oriented rows of discrete foci (white arrows) corresponding to the interface between A and I bands as determined by imaging the same field by confocal microscopy (d) and by phase contrast microscopy (e) (white arrows). Intensity of labeling at the cell surface (d) was indistinguishable from background (arrowheads). Magnifications: a and b, times 550; c, times 1,400; d and e, times 1,040; f, times 1,750. Scale bar, 10 µm.



cDNA Sequence Determination

A canine cardiac gt11 expression library (11) was screened with affinity-purified antibodies to the intact protein and to peptide 1. Two clones were isolated (clones 40 and 46), which reacted with antibody raised to the intact protein (Fig. 7). Although clone 40 bound antibody to peptide 1, clone 46 did not and was later shown to be missing this part of the protein coding region. A synthetic oligonucleotide probe corresponding to the first 30 bp of the 5`-untranslated region of clone 40 was used to screen a canine cardiac gt10 cDNA library(42) , and full-length clone 2 was isolated, which overlapped completely clones 40 and 46. We also obtained two additional clones (6 and 8) from a canine skeletal muscle cDNA expression library by screening with antibody to the intact protein. All of the clones depicted in Fig. 7were sequenced in both directions and were identical in the overlapping regions.

The nucleotide sequence of cardiac clone 2 is depicted in Fig. 8. It contained 80 bp of 5`-untranslated sequence, 633 bp of protein coding sequence including start and stop codons, and 1099 bp of 3`-untranslated sequence containing two consensus polyadenylation signals (AATAAA) (51) and a poly(A) tail. The ATG start codon depicted in Fig. 8was in the proper nucleotide setting for translation initiation(52) . This observation coupled with the N-terminal amino acid sequence obtained from the intact protein (residues 2-18 underlined in Fig. 8) established the translation initiation site at the first in-frame ATG. The 26-kDa CSQ binding protein, like triadin(16) , was missing the N-terminal methionine, suggesting that post-translational processing may occur. A total of 86 sequenced amino acid residues were found within the deduced sequence, confirming the identity of the clone (Fig. 8, underlined). Clone 8 from skeletal muscle, which encoded the full-length protein, also contained a poly(A) tail but was approximately half as long as cardiac clone 2 (Fig. 7). The poly(A) tail of clone 8 began 18 bp downstream from the internal polyadenylation signal. Northern blot analysis revealed two major mRNA transcripts of approximately 0.9 and 1.8 kilobases in both cardiac and skeletal muscle (Fig. 9), sizes predicted from the inserts in clones 8 and 2, respectively. These two mRNAs may result from alternative use of the two polyadenylation signals. Based on these results and the immunoblot analyses described above, we conclude that the same gene encodes identical 26-kDa CSQ binding proteins in heart and skeletal muscle. For the remainder of this paper we refer to this protein as Junctin, denoting a major CSQ binding protein of the junctional SR membrane in both cardiac and skeletal muscle.


Figure 9: Northern blot analysis. 30 µg of total RNA prepared from canine cardiac (C) and skeletal (S) muscle were analyzed. Filled and open arrowheads indicate the 900- and 1,800-bp species, respectively.



Protein Sequence Analysis

The cDNA sequence of Junctin predicts a protein of 210 amino acids with a calculated molecular weight of 23,496. This molecular weight is similar to value of 26,000 obtained by SDS-PAGE. Amino acid and nucleotide sequencing results did not provide an explanation for the migration of the protein as a doublet on SDS-PAGE. Partial proteolysis or post-translational modifications are possibilities but require further investigation. The protein did not appear to be glycosylated, as it contained no consensus glycosylation sites and was not retained by lectin affinity columns. Hydropathy analysis (Fig. 10) predicts that Junctin has a single transmembrane domain extending from residue 23 to 44. This region of the molecule contains a high density of hydrophobic amino acids and no charged residues. Several positively and negatively charged residues are concentrated near the N- and C-terminal sides of the transmembrane region, respectively. This type of charge polarization near the transmembrane domain predicts that the protein is synthesized with residues 1-22 facing the cytoplasm and residues 45-210 located in the SR lumen(53) . The absence of a signal peptide sequence also suggests that the N-terminal portion of the molecule faces the cytoplasm.


Figure 10: Hydropathy plot of 26-kDa CSQ binding protein. The graph was constructed using a window size of 19 amino acid residues(60) . Negative values reflect hydrophobicity.



A remarkable feature of Junctin is its very high density of charged residues (99 of 210 or 47.1%). The protein has a calculated pI of 9.37 with an excess of 17 basic residues over acidic residues. The lumenal portion of the molecule (residues 45-210) contains 54.8% charged residues with a calculated pI of 9.31. Notable in this region of the molecule is the frequent occurrence of two to three consecutive lysine or arginine residues situated between small clusters of acidic residues. The protein contains no cysteine residues, and we observed that its mobility on SDS-PAGE was unaffected by reducing agents (data not shown).

A search of the ENTREZ data base (Release 18, August 15, 1995) revealed significant homologies with liver ER aspartyl beta-hydroxylase (54) and rabbit skeletal muscle triadin(16) . Residues 4-76 of Junctin overlapped nearly perfectly with the sequence of bovine aspartyl beta-hydroxylase between amino acids 38 and 125 (Fig. 11). Of the 73 amino acids of junctin aligning with this region, 70 were identical to those of aspartyl beta-hydroxylase. One long run of 43 consecutive amino acids matched exactly. The sequence overlap encompassed most of the N-terminal residues of Junctin, extended through its transmembrane domain, and ended 32 residues into the lumenal region. Beyond this region, the proteins diverged. An insertion of 15 amino acids in aspartyl beta-hydroxylase (residues 88-102) divided the homology region in two, but a recently reported clone from human osteosarcoma cells (55) did not have the insertion, making the sequence overlap even more striking. Edman degradation of Junctin (Fig. 8) substantiated many of the identical amino acid residues. Further characterization will be required to determine if the two proteins are the products of the same or different genes.

The data base search also revealed small regions of sequence similarity with triadin. The amino acid sequence of triadin from residue 45 to 73 was 62% identical to residues 21-49 of Junctin (Fig. 11). This sequence similarity covered most of the transmembrane regions and extended five residues into the lumenal sequences ending with the identical amino acids DLVDY. Like Junctin, triadin is basic and has a high density of charged amino acids situated in the SR lumen(16) . Several short runs of identical sequences were detected here. For example, the sequence of amino acid residues 101-105 (KEKEK) of Junctin was repeated four times in the triadin sequence. Residues 91-96 (KELTKE) of Junctin were identical to residues 472-477 of triadin; residues 144-148 (PKGKK) of Junctin were the same as residues 392-396. Several other short runs of sequence similarity, composed mostly of charged residues, were also noted. Other features shared between the two proteins are considered under ``Discussion.''

Topological Analysis

We used protease treatment of intact and permeabilized junctional SR vesicles, coupled with immunoblotting, to ascertain the membrane topology of Junctin. The antibodies used for the analysis (to the intact protein, peptide 1, and peptide 2) recognized the predicted lumenal domain of the molecule. (The antibody produced to the intact protein recognized the lumenal region. It was used for detection of clones 6 and 46, which did not encode the N-terminal residues.)

When intact cardiac junctional SR vesicles were exposed to trypsin and blotted, the antibody staining intensity of the Junctin doublet was unchanged, but its mobility was increased by approximately 2 kDa (Fig. 12). This is consistent with cleavage by trypsin at arginine 14 or lysine 15, which would account for the observed mobility shift. All lysine and arginine residues beyond residue 15 were predicted to be located inside the SR vesicles and protected by the membrane from trypsin. The similar mobility shift and spacing between the two bands of the doublet after trypsin treatment suggested that both bands of the doublet had lost equivalent amounts of cytoplasmic amino acids and supported the notion that the proteins comprising the two bands were the same. When junctional SR vesicles were proteolyzed in the presence of 0.2% Triton X-100, all immunoreactivity was lost (Fig. 12). This was consistent with loss of the membrane-protected compartment and digestion of the intralumenal epitopes.


Figure 12: Trypsin digestion of canine cardiac SR vesicles. Membranes were incubated without trypsin (lane 1) or with trypsin at a 1/20 weight ratio in the absence (lane 2) or presence (lane 3) of 0.2% Triton X-100. Samples were electrophoresed and transferred to nitrocellulose, and blots were analyzed with antibodies to the intact protein (A), Peptide 1 (B), and Peptide 2 (C). Filled and open arrowheads indicate the intact and N-terminally cleaved 26-kDa protein doublet, respectively.




DISCUSSION

In this study we have purified and cloned a major integral membrane protein of junctional SR in cardiac and skeletal muscle. The protein analyzed, Junctin, is likely to play an important role in excitation-contraction coupling in both tissues, based on its high content in the junctional SR membrane, its co-localization with ryanodine receptors, and its ability to bind CSQ.

The topology of Junctin, consisting of only 210 amino acids, is relatively simple (Fig. 13). The protein contains a single transmembrane domain extending from residue 23 to 44. The first 22 amino acids are located in the cytoplasm, and the bulk of the molecule, composed of residues 45-210, is oriented in the SR lumen. A notable feature of Junctin is its basic character and high concentration of charged amino acids inside the SR compartment. This high charge density may be important for Junctin's ability to bind CSQ (which also is highly charged and acidic) (11) and also explains the substantial purification achieved by phosphocellulose chromatography. Comparison of the properties of Junctin with another recently cloned integral SR protein, triadin(16) , is particularly revealing. Both proteins bind CSQ and are selectively localized to junctional SR in cardiac and skeletal muscle(14, 15, 18, 49, 56) . The membrane topologies and overall structures are similar(16) . Both proteins span the SR membrane once, have short N-terminal sequences facing the cytoplasm, and project highly charged (50% charged residues), basic domains into the SR lumen. Sequence homologies are apparent, and neither protein contains a cleaved signal sequence(16) . Although skeletal muscle triadin is larger than Junctin (706 versus 210 amino acids), cardiac isoforms of triadin have recently been sequenced, which are approximately half the size of skeletal muscle triadin.^2 Two of these isoforms were detected here as CSQ binding proteins in cardiac SR vesicles ( Fig. 1and Fig. 2), but they bound much less CSQ than did Junctin. In contrast to skeletal muscle triadin, Junctin does not appear to be glycosylated and does not form disulfide-linked oligomers. However, cardiac triadin also does not oligomerize(15) .^2 Although disulfide bond formation by skeletal muscle triadin has been implicated in a functional role(57) , this cannot be the case for Junctin, which contains no cysteine residues.


Figure 13: Topology of Junctin in SR membrane. The N terminus (NH(2)) is oriented outside (Cytoplasm) and the C terminus (COOH) inside (Lumen). One transmembrane domain (Membrane) connects these two regions. Filled circles indicate residues identical with triadin. The region of sequence match with aspartyl beta-hydroxylase, where 70 of 73 residues are identical, is delimited by the large arrowheads. Pluses and minuses denote positively and negatively charged amino acids, respectively.



A striking homology was noted between Junctin and the ER enzyme aspartyl beta-hydroxylase(54, 55) . The amino acid sequences of the two proteins were virtually identical over 73 residues, including 19 residues of cytoplasmic sequence, the transmembrane regions, and extending 32 residues into the lumenal compartment, at which point the sequences diverged completely (Fig. 13). Aspartyl beta-hydroxylase contains 629 amino acids beyond the point of divergence, which are predicted to be entirely intralumenal and to contain the catalytic site (54) . It has been proposed that a type II signal anchor domain (i.e. the transmembrane domain) of aspartate beta-hydroxylase functions to direct the catalytic domain into the ER lumen(54) . Junctin, triadin, and aspartate beta-hydroxylase are most similar in sequence around the transmembrane domains (Fig. 13), and it is possible that the primary role of the signal anchors in all three proteins is to target the functionally distinct C-terminal regions into the ER interior. It may be that all three proteins evolved from one or two ancestor genes, but testing this idea will require further investigation.

The function of Junctin remains to be determined, but it appears to fulfill many of the requirements to be a major CSQ binding protein in vivo. Immunofluorescence and immunoblotting results demonstrate that it co-localizes with CSQ and ryanodine receptors, and it is abundantly expressed in both heart and skeletal muscle. Use of the nitrocellulose blotting method has shown Junctin to be the major CSQ binding protein in junctional SR vesicles (19) (Fig. 1). While native protein-protein interactions could be perturbed using our I-CSQ binding assay, the same technique has identified two cardiac triadin isoforms, suggesting that the method is reliable. Moreover, in independent studies we have observed that Junctin solubilized from SR membranes in the non-denaturing detergent Triton X-100 binds selectively to a cardiac CSQ affinity matrix and that Junctin antibodies immunoprecipitate CSQ from Triton X-100 extracts of SR membranes. One puzzling discrepancy between laboratories is that we find that binding of CSQ to Junctin (as well as to cardiac triadin) is inhibited by Ca(19) , whereas Guo and Campbell (18) report that CSQ binding to skeletal muscle triadin is Ca-dependent. We obtain the same CSQ binding result after transfer of SR proteins to nitrocellulose (19) or by binding Junctin to a CSQ affinity matrix. Ca inhibition of I-CSQ binding to Junctin on nitrocellulose blots was confirmed by Damiani and Margreth(20) . We believe that the disparate results reported on Ca effects most likely are due to methodological differences between laboratories rather than reflecting real differences in the interactions between CSQ and triadin and Junctin.

The presence of a specific binding protein for CSQ in the junctional face membrane has long been suspected(8, 30, 58) . Rope-like fibers have been observed here by electron microscopy, which appear to anchor CSQ to the SR membrane(8, 13) . We earlier postulated that Junctin might be one such anchoring protein(19) . Recently, triadin has been proposed to be another(18) . The similar features of the two proteins suggest that either or both could participate in such an interaction. Costello et al.(8) observed that 10% of the CSQ adherent to the junctional face membrane remained firmly attached after extraction of the membranes with EDTA. Consistent with this observation, we observe that CSQ binds most strongly to Junctin when the interaction occurs in the absence of Ca. It remains to be determined if Junctin also binds to the ryanodine receptor, as has been reported for triadin(18) . Studies are currently in progress to test if this capability exists. A ternary complex between anchoring proteins, CSQ, and ryanodine receptors, however, does not appear to be required for association of CSQ with the junctional SR membrane. An ultrastructural analysis reveals that knock-out mice lacking ryanodine receptors appear to have normal amounts of electron-dense material, presumed to represent CSQ, targeted to the junctional SR region(59) . Junctin may be a part of a family of proteins enriched in the junctional SR membrane, which stabilize or bind other SR proteins important for Ca release. Identification of all of the components of this putative molecular complex is currently in progress and will be facilitated by our isolation of the cDNA clone for this major CSQ binding protein.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant HL28556 (to L. R. J.), by the Heart and Stroke Foundation of Ontario (to A. O. J.), and by the Medical Research Council of Canada (to A. O. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38414[GenBank].

§
To whom correspondence should be addressed: Krannert Inst. of Cardiology, Dept. of Medicine, 1111 W. 10th St., Indianapolis, IN 46202. Tel.: 317-630-6695; Fax: 317-630-8595.

(^1)
The abbreviations used are: SR, sarcoplasmic reticulum; bp, base pair(s); MOPS, 3-(N-morpholino)propanesulfonic acid; CSQ, calsequestrin; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis.

(^2)
Guo, W., Jorgensen, A. O., Jones, L. R., and Campbell, K. P.(1996) J. Biol. Chem., in press.


ACKNOWLEDGEMENTS

The technical assistance of Glenn Schmeisser in raising Junctin antibodies is appreciated. We thank Joyce Dwulet for amino acid sequencing and Wayne Arnold for expert technical assistance on immunofluorescence labeling. We also acknowledge Steve Cala for critically reading the manuscript and providing helpful comments.


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