- Email: [email protected]
Specific protein-protein interactions of calsequestrin with junctional sarcoplasmic reticulum of skeletal muscle
Vol. 172, No. 3, 1990 November
BIOCHEMICAL
15, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1253-1259
Specific protein-protein interactions junctional sarcoplasmic reticulum
of calsequestrin with of skeletal muscle
Ernest0 Damiani and Alfred0 Margreth’ Centro di Studio per la Biologia e la Fisiopatologia Muscolare, c/o lstituto di Patologia generale, Universita’ di Padova, Via Trieste 7535121 Padova, Italy Received
September
24,
1990
SUMMARY: Minor protein components of triads and of sarcoplasmic reticulum (SR) terminal cisternae (TC), i.e. 47 and 37 kDa peptides and 3130 kDa and 26-25 kDa peptide doublets, were identified from their ability to bind 1251 calsequestrin (CS) in the presence of EGTA. The CS-binding peptides are specifically associated with the junctional membrane of TC, since they could not be detected in junctional transverse tubules and in longitudinal SR fragments. The 31-30 kDa peptide doublet, exclusively, did not bind CS in the presence of Ca2+. Thus, different types of protein-protein interactions appear to be involved in selective binding of CS to junctional 0 1990Rcademlc Press,Inc. TC. Calsequestrin (CS) is the main lumenal, low-affinity high-capacity Ca2+binding protein of skeletal muscle sarcoplasmic reticulum (SR) (1). Its selective
localization
in the terminal cisternae (TC) of the SR (2) and
seemingly preferential binding to the junctional membrane domain, in which ryanodine-sensitive suggested
Ca2+-release
channels
are localized
(3), have
the existence of specific protein interactions and a possible
functional role of CS in Ca2+-release (4) in addition to Ca2+ storage within the SR (5). Current views
are divided between a direct interaction of CS with the
ryanodine receptor (RyR) on the lumenal side of TC membrane (6) and/or via CS-binding proteins in the 26 kDa-32 kDa range of molecular weights (7, 4). Electron microscopic observations favour
an indirect interaction of CS
with the junctional Ca 2+-release protein complex /foot structure of TC, through membrane-bound, anchoring protein filaments (8). Protein-protein interactions of CS with junctional SR
have been correlated with Ca2+-
dependent conformational changes of CS (7, 4) at a specific segment of the CS molecule (9). + To whom correspondence
and reprint requests should be addressed.
Abbreviations: CS: calsequestrin; JFM: junctional face membrane; MHC: myosin heavy chain; RyR: ryanodine receptor; SR: sarcoplasmic reticulum; TC: terminal cisternae; TT, transverse tubules. 0006-291X/90 1253
All
Copyright 6 1990 rights of reproduction
$1.50
by Academic Press. Inc. in any form resenvd.
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
In this study we used ligand blot protein-overlay
RESEARCH COMMUNICATIONS
techniques, and 125l-
labeled skeletal muscle CS, both rabbit and chicken CS, because of their shared fundamental properties along with species-specific differences (lo), in order to characterize tissue-specific patterns of CS-binding proteins and to demonstrate more conclusively that these proteins are confined to the junctional membrane domain of the SR.
MATERIALS
AND
METHODS
All chemicals were analytical grade and were obtained from Sigma Chemical Co. (St. Louis, MO), Merck A.G. (Darmstadt, Germany), or Carlo Erba (Milano, Italy). SR membranes from pure fast or slow (soleus and semitendinosus) muscles of adult male New Zealand albino rabbits, and from predominantly - fast back muscles of adult pigs, were subfractionated by the method of Saito et al. (1 l), as modified by lnui u. (12). Triads and junctional transverse tubule (TT) membranes were obtained as described by Salvatori .&& (13). The JFM was purified from TC, essentially as described by Costello u. (15) except that the detergent Ct2E8 was used instead of Triton X-100. Treatment of SR membranes with Tris-EDTA was carried out as described by Duggan and Martonosi (16). Purified CS from both rabbit and chicken muscle (10) was labeled with 1251, and tested for binding to membrane proteins, using the ligand blot assay described by Mitchell ti (6). SDS/pofyacrytamide gel electrophoresis was carried out according to Laemmli (17) using 7.5-15% polyacrylamide linear gradient gels. Slabs were stained with Coomassie blue. Apparent Mrs were calculated from a graph of relative mobilities versus log Mr of standard proteins (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 42.7 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozime, 14 kDa). Electrophoretic transfer of proteins onto nitrocellulose was carried out as reported previously (18). Autoradiography of blots was carried out at -80 C, using Beta-max films (Amersham, U.K.).
RESULTS
AND
DISCUSSION
Figure 1, a (lane 1) shows the Ponceau-red stained pattern of blotted proteins from a Laemmli’s slab gel of isolated junctional TC from rabbit fasttwitch muscle, revealing the presence of the RyR monomer and of a characteristically
prominent CS band, relative to the ATPase band (11). Ligand blots performed in parallel with t25l-labeled CS, in the presence of EGTA, demonstrated that several minor components of TC, corresponding to 47 and 37 kDa peptides, and to peptide doublets at 31-30 kDa and 26-25 kDa, respectively, are able to interact with CS. This fundamental pattern appeared also to be qualitatively the same, independent of whether CS from chicken pectoralis, or from rabbit fast muscle, was used as the ligand (Fig. 1, a, lanes 2, 4), implying the involvement of some common structural features of the two distinct forms of CS (10) in their EGTA-resistant, specific proteinprotein interactions. 1254
Vol.
172,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0b
0a 4
5
Molecular mass (KDa)
1
2
3
Molecular mass (KDa) I 200
97
ATPase,
97
66 66 43
CaC12 EGTA
+
+ -
+
31
43
21
31
+
21
Fiaure 1. Overlay of SR and TT-membrane proteins fast-twitch skeletal muscle with 12%Calsequestrin.
from
rabbit
a) 60 pg of junctional TC proteins were electrophoresed on SDS 7.515% polyacrylamide linear gradient gels and electroblotted onto nitrocellulose. Lane 1 was stained with Ponceau red. Lanes 2 and Zwere incubated with 125l-labeled CS prepared from chicken pectoralis muscle, and lanes 4 and 5 with CS from rabbit fast muscles. Incubation was carried out for 60 min at room temperature in a medium having composition identical to that described by Mitchell u. (7) and containing 0.33 pg per ml of 125llabeled CS and either 1 mM EGTA or 1 mM CaCl2,as indicated. Blots, after washing, were dried and autoradiographed for 72 h (see Methods). b) Isolated triads (lane l), LSR fragments (lane 2) and purified junctional TT (lane 3) were analyzed by electrophoresis as in a (about 60 pg protein loaded per lane ), blotted and incubated with 125l-labeled chicken CS in the presence of 1 mM EGTA. RyR: ryanodine receptor (apparent Molecular mass 350 kDa in the specified electrophoretic conditions); CS: calsequestrin (64 kDa).
As compared with junctional TC, only isolated triads (Fig.1 b, lane l), but not either LSR fragments (Fig.1 b, lane 2) or junctional TT (Fig.1 b, lane 3), interacted with 1251-labeled CS, thus localizing the specific CS-binding proteins to the junctional membrane domain of TC. None of the CS-binding peptides could be solubilized from TC, either by using detergent C12Eg in the presence of Ca2+ (Fig.2, a), or by treatment with Tris-EDTA at alkaline pH (Fig. 2 b). The consequent coenrichment in these peptides and in RyR of the purified JFM-CS complex, and in CSdepleted vesicles, respectively, thus argues for a tight binding to membrane of 125l-CS labeled peptides. Labeling studies with photoactivatable phospholipid analogs (19) had similarly identified integral proteins in junctional SR of rabbit skeletal muscle, of 49, 37, 32 and 30 kDa, respectively. 12.55
Vol. 172, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0b
0a Molecular mass (kDa)
I
Molecular mass (kDa)
2
-
1
2
3
-RyR
4
-RyR
! I
43-
31.. 21.
Fia. 2. 12514X ligand blots of SDS gels of JFM proteins and of junctional TC subfractions after treatment with EDTA at alkaline PH. a) JFM was purified from junctional TC as a JFM-CS complex, using the detergent Ct2EB and 1 mM Ca2+. 60 ug of proteins were electrophoresed as in Fig. 1 in duplicate. The ligand blot assay was carried out with t25llabeledchicken CS, in the presence of 1 mM EGTA. Lane 1: autoradiogram; Lane 2: slab gel after stainingwith Coomassieblue. b) TC membraneswere incubated with 1 mM EDTA at alkaline pH (17) and centrifuged to obtain a pellet (Lanes 1 and 3) and a supernatant (lanes 2 and 4). 60 ug of proteins were electrophoresedas in Fig. 1. Lanes 3 and 4 (slab gels) were stained with Coomassie blue. lanes 1 and 2 were incubated with 126l-labeled chicken CS and electroblotted, autoradiographedas in Fig. 1.
We investigated the effect of Ca*+ on the interaction of 1*%labeled
CS
with junctional SR proteins. As shown in Fig. I, a, the presence of Ca*+ in the ligand blot assay specifically inhibited the binding of 1*%labeled CS to the 31-30 kDa protein doublet. Labeling studies of the purified JFM-CS complex with a covalently
reactive, thiol-specific
fluorescent
probe, had
similarly identified protein components of 31 and 29 kDa, in addition to the RyR, in relation to Ca *+-dependent fluorescence changes that required the presence of attached CS (4). The ubiquitous presence of about 30 kDa peptide doublets in junctional TC of pure fast and slow muscle of the rabbit (Fig. 3 A), and of mixed muscles of the pig (Fig. 3 B), provides an indication of their skeletal muscle specificity, independent of fiber types and of the animal species. The probably homologous 26 kDa peptide described by Mitchell et al. (7) in 1256
Vol.
172,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
0
RESEARCH
COMMUNICATIONS
0
A
B
1
Molecular mass(kDa)
2
4
3
97 f36-
u. Comparative electrophoretic and ligand blot analysis of isolated junctional TC from fast-twitch and slow-twitch muscle of the rabbit, and from pig back muscles. A) 60 pg of TC protein were electrophoresed as in Fig. 1. Lanes 1 and 2: Ponceau red-stained gels. Lanes 3 and 4: autoradiograms of blots after incubating with 12% labeled rabbit CS, in the presence of 1 mM EGTA. Fast-muscle TC: Lanes 2 and 4. Slow-muscle TC: Lanes 1 and 3. In autoradiograms, CS is detectable as the negative image of coincident Ponceau-red stained protein bands, corresponding to the fast-skeletal (CSf, 64 kDa) and slow-cardiac (CSc, 54 kDa) isoform of CS, respectively. B) 60 pg of TC proteins were electrohoresed and blotted onto nitrocellulose, as described in the legend to Fig. 1. I ane 1 was stained with Ponceau red. Lanes 2 and 3 were incubated with 125Habeled CS, prepared from rabbit fast muscle, in the presence of 1 mM EGTA. Lanes 1 and 2: TC from mixed, predominantly fast muscles of adult pig; Lane 3: TC from rabbit slow muscle.
junctional
SR of dog heart muscle,
exhibiting
an identical
interaction
with CS, might therefore
correspond
Ca*+-dependent
to the cardiac
isoform
of the
same protein (s). EGTA-resistant
protein-protein
interactions
of CS with junctional
which,
unlike that with the 31-30 kDa peptide doublet,
Ca*+,
and, therefore,
are not related
changes
of CS (4,7),
molecule,
recently identified by Collins u.
The presence
need
to Ca*+-induced
not involve
the specific
SR and
are not affected
by
conformational region
of the CS
protein-binding
site, such
(9).
in CS of more than one potential
as a high number of negatively charged amino acids (5), would also be consistent with an ionic type of interaction involving these groups, and positively 1*5Habeled
charged
groups
on the interacting
CS binds strongly
to lysozyme
proteins.
We have found that
on ligand blots in the presence
of EGTA. Costello .@& (15) reported that after treatment with EDTA about 10% of CS remained tightly bound to the JFM, and lkemoto et. (4) showed that binding of CS to JFM is critically affected by ionic strength. 1257
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The existence of multiple molecular interactions of a group of junctional SR proteins, rather than a single protein (7), with CS, as well as, even though entirely speculative at the present time, between each other and with the RyR,
are all possibilities
interconnected,
that
deserve
attention
in relation
to two
and still open problems, i.e., a) the postulated regulatory
role of CS on SR Ca*+-release (4), and b) the precise mode of membrane attachment and the intrinsic composition of the anchoring protein filaments, morphologically described in TC by Franzini-Armstrong g& (8). Although it was shown that the Ca*+-release
properties of fast-twitch and slow-twitch
muscle fibers are heterogeneous (20), that does not appear to reflect the existence of distinct isoforms of the RyR (21, 22). In this connection, it may be significant that ligand blot analysis of TC components interacting with CS in a Ca*+-independent
manner, (Fig. 3, A and B), revealed lesser differences
from the characteristic mixed, predominantly
pattern seen in rabbit fast muscle, in the case of fast muscle of the pig, as compared with TC from
rabbit slow muscle, lacking completely the 26-25 kDa peptide doublet.
Acknowledgments.
This work supported by funds from the Consiglio Nazionale delle Ricerche and by grants from Minister0 Pubblica lstruzione to A.M. The technical help of Mr G.A. Tobaldin is gratefully acknowledged.
REFERENCES 1. Campbell, K.P. (1986) in Sarcoplasmic Reticulum in Muscle Physiology (Entmann, M.L. & Van Winkle, B., eds.), pp. 65-69, CRC Press, Boca Raton, FL. 2. Jorgensen, A.O. (1987) Amer. Zool. 27, 1021-1032. 3. Fleischer, S. and Inui, M. (1989) Ann. Rev. Biophys. Biophys. Chem. 18, 333-364. 4. lkemoto, N., Ronjat, M., Meszaros, L.G. and Koshita, M. (1989) Biochemistry 28, 6764-6771. 5. MacLennan, D.H. and Wong, P.T.S. (1971) Proc. Natl. Acad. Sci. USA 68, 1231-1235. 6. Marks, A.R., Fleischer, S. and Tempst, P. (1990) J. Biol. Chem. 265, 13143-l 3149. 7. Mitchell, R.D., Simmerman, H.K.B. and Jones, L.R. (1988) J. Biol. Chem. 263, 1376-l 381. 8. Franzini-Armstrong, C., Kenney, L.J. and Varriano-Marston (1987) J. Cell Biol. 105, 49-56. 9. Collins, J.H., Tarcsafalvi, A. and Ikemoto, N. (1990) Biochem. Biophys. Res. Comm. 167, 189-193. 10. Damiani, E., Salvatori, S. and Margreth, A. (1990) J. Must. Res. Cell Motil. 11, 48-56. Il. Saito, A., Seiler, S., Chu, A. and Fleischer, S. (1984) J. Cell Biol. 99, 875 885. 12. Inui, M., Saito, A. and Fleischer, S. (1987) J. Biol. Chem. 262, 17401747. 1S.Salvatori, S., Damiani, E., Barhanin, J., Furlan, S., Salviati, G. and Margreth, A. (1990) Biochem. J. 267, 679-687. 1258
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14. Lowry, O.H., Rosebrough, N.J., Fan, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 15. Costello, B., Chadwick, C., Saito, A., Chu, A., Maurer, A. and Fleischer, S. (1986) J. Cell Biol. 103, 741-753. 16. Duggan, P.F. and Martonosi, A. (1970) J. Gen. Physiol. 46, 147-l 57. 17. Laemmli, U.K. (1970) Nature (London) 227, 680-685. 18. Damiani, E., Spamer, C., Heilmann, C., Salvatori, S. and Margreth, A. (1988) J. Biol. Chem. 263, 340-343. 19. Volpe, P., Gutweniger, H. and Montecucco, C. (1987) Arch. Biochem. Biophys. 253, 138-l 45. 20. Salviati, G. and Volpe, P. (1988) Am. J. Physiol. 254, C459-465. 21. Damiani, E., Volpe, P. and Margreth, A. (1990) J. Must. Res. Cell Motil., in press. 22.0tsu, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.M. and MacLennan, D.H. (1990) J. Biol.. Chem. 265, 13472-13483.
1259
BIOCHEMICAL
15, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1253-1259
Specific protein-protein interactions junctional sarcoplasmic reticulum
of calsequestrin with of skeletal muscle
Ernest0 Damiani and Alfred0 Margreth’ Centro di Studio per la Biologia e la Fisiopatologia Muscolare, c/o lstituto di Patologia generale, Universita’ di Padova, Via Trieste 7535121 Padova, Italy Received
September
24,
1990
SUMMARY: Minor protein components of triads and of sarcoplasmic reticulum (SR) terminal cisternae (TC), i.e. 47 and 37 kDa peptides and 3130 kDa and 26-25 kDa peptide doublets, were identified from their ability to bind 1251 calsequestrin (CS) in the presence of EGTA. The CS-binding peptides are specifically associated with the junctional membrane of TC, since they could not be detected in junctional transverse tubules and in longitudinal SR fragments. The 31-30 kDa peptide doublet, exclusively, did not bind CS in the presence of Ca2+. Thus, different types of protein-protein interactions appear to be involved in selective binding of CS to junctional 0 1990Rcademlc Press,Inc. TC. Calsequestrin (CS) is the main lumenal, low-affinity high-capacity Ca2+binding protein of skeletal muscle sarcoplasmic reticulum (SR) (1). Its selective
localization
in the terminal cisternae (TC) of the SR (2) and
seemingly preferential binding to the junctional membrane domain, in which ryanodine-sensitive suggested
Ca2+-release
channels
are localized
(3), have
the existence of specific protein interactions and a possible
functional role of CS in Ca2+-release (4) in addition to Ca2+ storage within the SR (5). Current views
are divided between a direct interaction of CS with the
ryanodine receptor (RyR) on the lumenal side of TC membrane (6) and/or via CS-binding proteins in the 26 kDa-32 kDa range of molecular weights (7, 4). Electron microscopic observations favour
an indirect interaction of CS
with the junctional Ca 2+-release protein complex /foot structure of TC, through membrane-bound, anchoring protein filaments (8). Protein-protein interactions of CS with junctional SR
have been correlated with Ca2+-
dependent conformational changes of CS (7, 4) at a specific segment of the CS molecule (9). + To whom correspondence
and reprint requests should be addressed.
Abbreviations: CS: calsequestrin; JFM: junctional face membrane; MHC: myosin heavy chain; RyR: ryanodine receptor; SR: sarcoplasmic reticulum; TC: terminal cisternae; TT, transverse tubules. 0006-291X/90 1253
All
Copyright 6 1990 rights of reproduction
$1.50
by Academic Press. Inc. in any form resenvd.
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
In this study we used ligand blot protein-overlay
RESEARCH COMMUNICATIONS
techniques, and 125l-
labeled skeletal muscle CS, both rabbit and chicken CS, because of their shared fundamental properties along with species-specific differences (lo), in order to characterize tissue-specific patterns of CS-binding proteins and to demonstrate more conclusively that these proteins are confined to the junctional membrane domain of the SR.
MATERIALS
AND
METHODS
All chemicals were analytical grade and were obtained from Sigma Chemical Co. (St. Louis, MO), Merck A.G. (Darmstadt, Germany), or Carlo Erba (Milano, Italy). SR membranes from pure fast or slow (soleus and semitendinosus) muscles of adult male New Zealand albino rabbits, and from predominantly - fast back muscles of adult pigs, were subfractionated by the method of Saito et al. (1 l), as modified by lnui u. (12). Triads and junctional transverse tubule (TT) membranes were obtained as described by Salvatori .&& (13). The JFM was purified from TC, essentially as described by Costello u. (15) except that the detergent Ct2E8 was used instead of Triton X-100. Treatment of SR membranes with Tris-EDTA was carried out as described by Duggan and Martonosi (16). Purified CS from both rabbit and chicken muscle (10) was labeled with 1251, and tested for binding to membrane proteins, using the ligand blot assay described by Mitchell ti (6). SDS/pofyacrytamide gel electrophoresis was carried out according to Laemmli (17) using 7.5-15% polyacrylamide linear gradient gels. Slabs were stained with Coomassie blue. Apparent Mrs were calculated from a graph of relative mobilities versus log Mr of standard proteins (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 42.7 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozime, 14 kDa). Electrophoretic transfer of proteins onto nitrocellulose was carried out as reported previously (18). Autoradiography of blots was carried out at -80 C, using Beta-max films (Amersham, U.K.).
RESULTS
AND
DISCUSSION
Figure 1, a (lane 1) shows the Ponceau-red stained pattern of blotted proteins from a Laemmli’s slab gel of isolated junctional TC from rabbit fasttwitch muscle, revealing the presence of the RyR monomer and of a characteristically
prominent CS band, relative to the ATPase band (11). Ligand blots performed in parallel with t25l-labeled CS, in the presence of EGTA, demonstrated that several minor components of TC, corresponding to 47 and 37 kDa peptides, and to peptide doublets at 31-30 kDa and 26-25 kDa, respectively, are able to interact with CS. This fundamental pattern appeared also to be qualitatively the same, independent of whether CS from chicken pectoralis, or from rabbit fast muscle, was used as the ligand (Fig. 1, a, lanes 2, 4), implying the involvement of some common structural features of the two distinct forms of CS (10) in their EGTA-resistant, specific proteinprotein interactions. 1254
Vol.
172,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0b
0a 4
5
Molecular mass (KDa)
1
2
3
Molecular mass (KDa) I 200
97
ATPase,
97
66 66 43
CaC12 EGTA
+
+ -
+
31
43
21
31
+
21
Fiaure 1. Overlay of SR and TT-membrane proteins fast-twitch skeletal muscle with 12%Calsequestrin.
from
rabbit
a) 60 pg of junctional TC proteins were electrophoresed on SDS 7.515% polyacrylamide linear gradient gels and electroblotted onto nitrocellulose. Lane 1 was stained with Ponceau red. Lanes 2 and Zwere incubated with 125l-labeled CS prepared from chicken pectoralis muscle, and lanes 4 and 5 with CS from rabbit fast muscles. Incubation was carried out for 60 min at room temperature in a medium having composition identical to that described by Mitchell u. (7) and containing 0.33 pg per ml of 125llabeled CS and either 1 mM EGTA or 1 mM CaCl2,as indicated. Blots, after washing, were dried and autoradiographed for 72 h (see Methods). b) Isolated triads (lane l), LSR fragments (lane 2) and purified junctional TT (lane 3) were analyzed by electrophoresis as in a (about 60 pg protein loaded per lane ), blotted and incubated with 125l-labeled chicken CS in the presence of 1 mM EGTA. RyR: ryanodine receptor (apparent Molecular mass 350 kDa in the specified electrophoretic conditions); CS: calsequestrin (64 kDa).
As compared with junctional TC, only isolated triads (Fig.1 b, lane l), but not either LSR fragments (Fig.1 b, lane 2) or junctional TT (Fig.1 b, lane 3), interacted with 1251-labeled CS, thus localizing the specific CS-binding proteins to the junctional membrane domain of TC. None of the CS-binding peptides could be solubilized from TC, either by using detergent C12Eg in the presence of Ca2+ (Fig.2, a), or by treatment with Tris-EDTA at alkaline pH (Fig. 2 b). The consequent coenrichment in these peptides and in RyR of the purified JFM-CS complex, and in CSdepleted vesicles, respectively, thus argues for a tight binding to membrane of 125l-CS labeled peptides. Labeling studies with photoactivatable phospholipid analogs (19) had similarly identified integral proteins in junctional SR of rabbit skeletal muscle, of 49, 37, 32 and 30 kDa, respectively. 12.55
Vol. 172, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0b
0a Molecular mass (kDa)
I
Molecular mass (kDa)
2
-
1
2
3
-RyR
4
-RyR
! I
43-
31.. 21.
Fia. 2. 12514X ligand blots of SDS gels of JFM proteins and of junctional TC subfractions after treatment with EDTA at alkaline PH. a) JFM was purified from junctional TC as a JFM-CS complex, using the detergent Ct2EB and 1 mM Ca2+. 60 ug of proteins were electrophoresed as in Fig. 1 in duplicate. The ligand blot assay was carried out with t25llabeledchicken CS, in the presence of 1 mM EGTA. Lane 1: autoradiogram; Lane 2: slab gel after stainingwith Coomassieblue. b) TC membraneswere incubated with 1 mM EDTA at alkaline pH (17) and centrifuged to obtain a pellet (Lanes 1 and 3) and a supernatant (lanes 2 and 4). 60 ug of proteins were electrophoresedas in Fig. 1. Lanes 3 and 4 (slab gels) were stained with Coomassie blue. lanes 1 and 2 were incubated with 126l-labeled chicken CS and electroblotted, autoradiographedas in Fig. 1.
We investigated the effect of Ca*+ on the interaction of 1*%labeled
CS
with junctional SR proteins. As shown in Fig. I, a, the presence of Ca*+ in the ligand blot assay specifically inhibited the binding of 1*%labeled CS to the 31-30 kDa protein doublet. Labeling studies of the purified JFM-CS complex with a covalently
reactive, thiol-specific
fluorescent
probe, had
similarly identified protein components of 31 and 29 kDa, in addition to the RyR, in relation to Ca *+-dependent fluorescence changes that required the presence of attached CS (4). The ubiquitous presence of about 30 kDa peptide doublets in junctional TC of pure fast and slow muscle of the rabbit (Fig. 3 A), and of mixed muscles of the pig (Fig. 3 B), provides an indication of their skeletal muscle specificity, independent of fiber types and of the animal species. The probably homologous 26 kDa peptide described by Mitchell et al. (7) in 1256
Vol.
172,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
0
RESEARCH
COMMUNICATIONS
0
A
B
1
Molecular mass(kDa)
2
4
3
97 f36-
u. Comparative electrophoretic and ligand blot analysis of isolated junctional TC from fast-twitch and slow-twitch muscle of the rabbit, and from pig back muscles. A) 60 pg of TC protein were electrophoresed as in Fig. 1. Lanes 1 and 2: Ponceau red-stained gels. Lanes 3 and 4: autoradiograms of blots after incubating with 12% labeled rabbit CS, in the presence of 1 mM EGTA. Fast-muscle TC: Lanes 2 and 4. Slow-muscle TC: Lanes 1 and 3. In autoradiograms, CS is detectable as the negative image of coincident Ponceau-red stained protein bands, corresponding to the fast-skeletal (CSf, 64 kDa) and slow-cardiac (CSc, 54 kDa) isoform of CS, respectively. B) 60 pg of TC proteins were electrohoresed and blotted onto nitrocellulose, as described in the legend to Fig. 1. I ane 1 was stained with Ponceau red. Lanes 2 and 3 were incubated with 125Habeled CS, prepared from rabbit fast muscle, in the presence of 1 mM EGTA. Lanes 1 and 2: TC from mixed, predominantly fast muscles of adult pig; Lane 3: TC from rabbit slow muscle.
junctional
SR of dog heart muscle,
exhibiting
an identical
interaction
with CS, might therefore
correspond
Ca*+-dependent
to the cardiac
isoform
of the
same protein (s). EGTA-resistant
protein-protein
interactions
of CS with junctional
which,
unlike that with the 31-30 kDa peptide doublet,
Ca*+,
and, therefore,
are not related
changes
of CS (4,7),
molecule,
recently identified by Collins u.
The presence
need
to Ca*+-induced
not involve
the specific
SR and
are not affected
by
conformational region
of the CS
protein-binding
site, such
(9).
in CS of more than one potential
as a high number of negatively charged amino acids (5), would also be consistent with an ionic type of interaction involving these groups, and positively 1*5Habeled
charged
groups
on the interacting
CS binds strongly
to lysozyme
proteins.
We have found that
on ligand blots in the presence
of EGTA. Costello .@& (15) reported that after treatment with EDTA about 10% of CS remained tightly bound to the JFM, and lkemoto et. (4) showed that binding of CS to JFM is critically affected by ionic strength. 1257
Vol.
172, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The existence of multiple molecular interactions of a group of junctional SR proteins, rather than a single protein (7), with CS, as well as, even though entirely speculative at the present time, between each other and with the RyR,
are all possibilities
interconnected,
that
deserve
attention
in relation
to two
and still open problems, i.e., a) the postulated regulatory
role of CS on SR Ca*+-release (4), and b) the precise mode of membrane attachment and the intrinsic composition of the anchoring protein filaments, morphologically described in TC by Franzini-Armstrong g& (8). Although it was shown that the Ca*+-release
properties of fast-twitch and slow-twitch
muscle fibers are heterogeneous (20), that does not appear to reflect the existence of distinct isoforms of the RyR (21, 22). In this connection, it may be significant that ligand blot analysis of TC components interacting with CS in a Ca*+-independent
manner, (Fig. 3, A and B), revealed lesser differences
from the characteristic mixed, predominantly
pattern seen in rabbit fast muscle, in the case of fast muscle of the pig, as compared with TC from
rabbit slow muscle, lacking completely the 26-25 kDa peptide doublet.
Acknowledgments.
This work supported by funds from the Consiglio Nazionale delle Ricerche and by grants from Minister0 Pubblica lstruzione to A.M. The technical help of Mr G.A. Tobaldin is gratefully acknowledged.
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