(Received for publication, August 30, 1995)
From the
Previously we identified a protein of apparent M = 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
-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.
The technique of membrane subcellular fractionation has proven
to be invaluable for biochemical dissection of the molecular components
of the SR ()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) .
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
) and pellet (P
) fractions. P
was
extracted with 2% Triton X-100 plus 0.5 M NaCl to yield
secondary supernatant (S
) and pellet (S
) 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.
The asterisk denotes a minor CSQ binding band, which appears to be related to
the 26-kDa CSQ binding protein.
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.
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.
Figure 11:
Sequence alignment of aspartyl
-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.
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 [
H]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.
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, 600; b,
1,100; d and e,
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,
550; c,
1,400; d and e,
1,040; f,
1,750. Scale bar, 10 µm.
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.
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 -hydroxylase (54) and
rabbit skeletal muscle triadin(16) . Residues 4-76 of
Junctin overlapped nearly perfectly with the sequence of bovine
aspartyl
-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
-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
-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.''
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.
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.
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) .
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) 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
-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 -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
-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
-hydroxylase functions to
direct the catalytic domain into the ER lumen(54) . Junctin,
triadin, and aspartate
-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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38414[GenBank].