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Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions
EXPERIMENTALCELL
RESEARCH
188,312-315
(1990)
SHORT NOTE Restoration of Gap Junction-like Structure after Detergent Sdubilization of the Proteins from Liver Gap Junctions F. MAZET AND J. L. MAZET Luboratoire
de Physiologic
Compa&e (CNRS,
UAllZl),
Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and o&y1 glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoro isso Academic P-, Inc. ing a gap junction-like StrUCtUre.
INTRODUCTION Reconstitution of liver gap junctions would allow a determination of the elements involved in their activity. It implies the solubilization, the isolation, and the identification of the membrane proteins. By electrophoresis analysis, it has been shown that rat liver gap junctions contain several proteins, among which the major protein migrates at 27-28 kDa [l-3]. No detergent, except the denaturing sodium dodecylsulfate (SDS), has been found to solubilize the subunits of the liver gap junction plaques, probably because of particular properties of the gap junction aggregates. The junctional domain contains a large amount of cholesterol unaffected by detergents even when phospholipids are removed [ 1 J. Assuming that the junctional particle aggregates were maintained by cholesterol, we have shown that the gap junction structure could be disrupted by digitonin, a cholesterol chelator, and completely disorganized by the addition of octyl glucoside [4]. It has been reported that the junctional protein MP26 of the lens [5-91 and the cardiac gap junction protein [lo], which are easier to solubilize than the liver gap
0014-4827/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Universite’ Paris-&d,
Bit. 443,91405-Orsay,
France
junctions, could be reconstituted into liposomes to form channels. In the present report, we show that gap junction disorganization by a mixture of detergents is associated with the partial solubilization of the junctional proteins. The solubilized proteins are able to reform gap junction-like structures when reconstituted into liposomes. Some of this material has been presented in preliminary form [ 111. MATERIAL
AND METHODS
Rat liver intact gap junctions were isolated and incubated in a mixture of digitonin and octyl glucoside as already described [4]. The aggregated material was eliminated by centrifugation (1 h, 110,OOCg) after dilution with 1 vol of phosphate buffer. The supernatant was concentrated with Centricon 10 microconcentrators (Amicon) down to half its initial volume and used for reconstitution experiments. A mixture of lecithin (Sigma, Type IIS) and cholesterol (Sigma, standard for chromatography) in chloroform was evaporated under a stream of nitrogen to form a thin lipid film and then solubilized into the concentrated solution from Centricon 10 by vigourous stirring. The cholesterol-digitonin precipitate was eliminated by centrifugation. The octyl glucoside was removed by dialysis (Spectrapor 2, Spectrum Medical Industries) against a TRIS buffer (20 n&4 TRIS-HCI, 100 mM NaCl, 5 m&f EDTA, pH 7.3) at 4’C for at least 72 h. The sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 12.5% polyacrylamide slab gel according to Laemmli. Aliquots of each sample were solubilized in a 2% SDS buffer containing 5% 2-mercaptoethanol and heated at 37°C for several minutes. The gels were stained with a silver staining kit (Bio-Rad). Glycerol buffer was added to the reconstituted material at a final concentration of 25%. Small drops of the preparations were deposited on conventional Balzer’s carrier sample and then rapidly frozen in liquid Freon-22. Fracturing and replication were done using a Balzer’s BAF 301 apparatus in a conventional way. The replicas were cleaned in clorox, rinsed with distilled water, and observed with a Siemens IA electron microscope. The pellets of intact gap junction and insoluble material were processed for electron microscopy as described previously [4].
RESULTS After incubation of the gap junctions in the detergent mixture of digitonin and octyl glucoside, the insoluble 312
SHORT
313
NOTE
fracture plane goes through adjacent membranes, the pits on the upper membrane closely correspond to the particles within the lower membrane (Fig. 2D). DISCUSSION
FIG. 1. Electrophoretic profile of gap junctions at different stages of the experiment. The protein composition of the insoluble material after detergent action (lane A) is similar to that of isolated gap junctions (lane C). The protein composition of the solubilized fraction was assayed after reconstitution into liposomes (lane B). The band profile of lane B is similar to that of lanes A and C and exhibits the same major band at 28 kDa. Markers: phosphorylase B, 92.5 kDa; bovine serum albumine, 66.2 kDa; ovalbumine, 45 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor: 21.5 kDa; and lysozyme: 14.4 kDa.
part of the initial material was centrifugated (1 h, 110,OOOg).The pellet was amorphous, indicating that the native structure of gap junctions had been completely destroyed [4]. We compare the protein compositions of the pellet and of the supernatant with the intact isolated liver gap junctions (Fig. 1). The supernatant was reconstituted into liposomes before SDS-PAGE analysis. The presence of proteins in the reconstituted sample (Fig. 1, lane B) proves that proteins have been solubilized by the detergent mixture although the solubilization was incomplete as indicated by the content of the pellet sample (Fig. 1, lane A). At all the stages of the experiment, the band pattern exhibits the major 27- to 2%kDa protein constituent of the liver gap junctions and a less important band of higher molecular weight. The similar electrophoresis profile of soluble and insoluble material to the intact gap junctions suggests that the detergent action is not selective. The solubilized fraction was reconstituted into liposomes at two different protein-to-lipid ratios. At low protein-to-lipid ratio we found the liposomes to be unilamellar vesicles with a small average diameter of 57 nm on freeze-fracture replicas. On the fracture faces of isolated liposomes, clusters of particles were often observed (Fig. 2B). When the vesicles were aggregated into pairs, a few particles appeared localized in the contact area (Fig. 2A). At high protein-to-lipid ratio (Figs. 2C and 2D) the density of particles within the fractures faces is greater and the reconstituted material may contain various types of vesicles in a single preparation. Aggregated particles in disordered arrays are visible on multilamellar liposomes (Fig. 2C). Large unilamellar vesicles carry randomlv dispersed particles of 7 to 12 nm. When the
The present results show that aggregates of particles are present in the vesicles reconstituted from solubilized liver gap junctions. The high-speed centrifugation (1 h, 110,OOOg)step ensures that these aggregates were absent from the starting material for reconstitution but were formed either at the time of the lipid addition or during the process of detergent removal. Two characteristics of the particle aggregates may be compared to the intact gap junctions. The association of two connexons from adjacent membranes seems to be recovered. This may be attributed both to a nondenaturing action of the detergents used and to the detergent removal by dialysis, a smooth method for reconstituting a lipid bilayer around solubilized membrane proteins. The aggregates formed in the plane of the membrane are reminiscent of in situ gap junctions. The requirement of digitonin to disrupt the gap junction structure suggests that cholesterol is involved in the junction assembly. This is consistent with the finding that the gap junction assembly between reaggregating Novikoff hepatoma cells depends on the cholesterol content of the culture medium [12]. The question arises why aggregates can be reconstituted in the presence of digitonin since, at every protein-to-lipid ratio used here, the added cholesterol should be chelated by the excess of digitonin. Assuming that the affinity of the cholesterol for gap junctions is of the same order of magnitude as that of the cholesterol-digitonin complex, one could explain both the incomplete solubilization and the possibility of reconstitution of particle assemblies. The protein composition of the reconstituted liposomes is identical to that of the intact isolated gap junctions. Characteristically, in both cases, the major proteins migrate at 27-28 kDa and a higher weight band is present. This suggests that the action of the detergents is not selective and that all the subunits present in the intact isolated gap junctions are spontaneously reincorporated into a gap junction-like structure. Aside from the restoration of the permeability and electrical activity properties of gap junctions, the characteristic assembly of particles is the best assay for functional preservation. In vitro channel activity has already been reported for the 27- to 2%kDa subunit solubilized from liver with SDS [13]. But without checking the structure restoration it is not easy to evaluate the degree of reconstitution. The present report is considered a first and necessary step toward the full reconstitution of gap junction activity.
314
SHORT
NOTE
FIG. 2. Solubilized fraction reconstituted into liposomes at two different protein concentrations. At low protein-to-lipid ratio, clusters of few particles are visible in the contact area between two vesicles (A: arrow) and on one side of single vesicles (B). Bar: 43.5 nm. At high proteinto-lipid ratio, the reconstituted material contains several kinds of vesicles. In multilamellar liposomes (C), the particles tend to aggregate into disordered arrays (bar: 128 nm). On another micrograph of the same preparation (D), the single arrow indicates a vesicle with a piece of adherent membrane where pits correspond with the particles of the vesicle membrane. The double arrows indicates the presence of pits and particles on the fracture face of a single vesicle. Bar: 109 nm.
We thank Dr. E. L. Benedetti and Dr. I. Dunia for providing helpful facilities to work in their laboratory (Inst. J. Monod, Univ. Paris VII, Paris) and helpful discussion and M. Recouvreur for his technical assistance. REFERENCES 1.
Henderson, 193-218.
D., Eibl, H., and Weber, K. (1979) J. Mol. Bid. 132,
2. 3.
4. 5.
Hertzberg, E. L., and Gilula, N. B. (1979) J. Biol. Chem. 264, 2138-2147. Nicholson, B. J., Hukapiller, M. W., Grim, L. B., Hood, L. L., and Revel, J.-P. (1981) Proc. N&l. Acad. Sci. USA 78, 75947598. Mazet, F., and Blattmann, A. (1988) C. R. Acad. Sci. Paris 307, 679-684. Gooden, M., Rintoul, D., Takekana, M., and Takemoto, L. (1985) Biochem. Biophys. Res. Commun. 128,993-999.
SHORT 6.
10.
Claassen, D. E., and Spooner, B. S. (1988) Bit&em. Biophys. Res.
M. (1985) Proc. N&l.
11.
Sci. USA 82,846~~8472. C. (1985) J. Memb. Bid. 83, 217-
12.
Mazet, F., and Mazet, J.-L. (1989) Molecular and Cell Biology of Gap Junctions, July 18-23, Irsee, FRG. Meyer, R., Malewicz, B., Baumann, W., and Johnson, R. G. (1987) J. CellBiol. 105,228a.
Nikaido,
H., and Rosenberg, E. Y. (1985) J. Membr. Bid. 86,87-
92. 7.
Commun. 154,194-198.
Zampighi,
Ad. 8.
G. A., Hall, J. E., and Kreman,
Girsch, S. J., and Peracchia,
225. 9.
315
NOTE
Dunia, I., Manenti,
S., Rousselet, A., and Benedetti,
J. Cell Biol. 105,1679-1689. Received October 2,1989
E. L. (1987)
13.
Young, J. D. E., Cohn, D. A., and Gilula,
733-743.
N. B. (1987) Cell 48,
RESEARCH
188,312-315
(1990)
SHORT NOTE Restoration of Gap Junction-like Structure after Detergent Sdubilization of the Proteins from Liver Gap Junctions F. MAZET AND J. L. MAZET Luboratoire
de Physiologic
Compa&e (CNRS,
UAllZl),
Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and o&y1 glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoro isso Academic P-, Inc. ing a gap junction-like StrUCtUre.
INTRODUCTION Reconstitution of liver gap junctions would allow a determination of the elements involved in their activity. It implies the solubilization, the isolation, and the identification of the membrane proteins. By electrophoresis analysis, it has been shown that rat liver gap junctions contain several proteins, among which the major protein migrates at 27-28 kDa [l-3]. No detergent, except the denaturing sodium dodecylsulfate (SDS), has been found to solubilize the subunits of the liver gap junction plaques, probably because of particular properties of the gap junction aggregates. The junctional domain contains a large amount of cholesterol unaffected by detergents even when phospholipids are removed [ 1 J. Assuming that the junctional particle aggregates were maintained by cholesterol, we have shown that the gap junction structure could be disrupted by digitonin, a cholesterol chelator, and completely disorganized by the addition of octyl glucoside [4]. It has been reported that the junctional protein MP26 of the lens [5-91 and the cardiac gap junction protein [lo], which are easier to solubilize than the liver gap
0014-4827/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Universite’ Paris-&d,
Bit. 443,91405-Orsay,
France
junctions, could be reconstituted into liposomes to form channels. In the present report, we show that gap junction disorganization by a mixture of detergents is associated with the partial solubilization of the junctional proteins. The solubilized proteins are able to reform gap junction-like structures when reconstituted into liposomes. Some of this material has been presented in preliminary form [ 111. MATERIAL
AND METHODS
Rat liver intact gap junctions were isolated and incubated in a mixture of digitonin and octyl glucoside as already described [4]. The aggregated material was eliminated by centrifugation (1 h, 110,OOCg) after dilution with 1 vol of phosphate buffer. The supernatant was concentrated with Centricon 10 microconcentrators (Amicon) down to half its initial volume and used for reconstitution experiments. A mixture of lecithin (Sigma, Type IIS) and cholesterol (Sigma, standard for chromatography) in chloroform was evaporated under a stream of nitrogen to form a thin lipid film and then solubilized into the concentrated solution from Centricon 10 by vigourous stirring. The cholesterol-digitonin precipitate was eliminated by centrifugation. The octyl glucoside was removed by dialysis (Spectrapor 2, Spectrum Medical Industries) against a TRIS buffer (20 n&4 TRIS-HCI, 100 mM NaCl, 5 m&f EDTA, pH 7.3) at 4’C for at least 72 h. The sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 12.5% polyacrylamide slab gel according to Laemmli. Aliquots of each sample were solubilized in a 2% SDS buffer containing 5% 2-mercaptoethanol and heated at 37°C for several minutes. The gels were stained with a silver staining kit (Bio-Rad). Glycerol buffer was added to the reconstituted material at a final concentration of 25%. Small drops of the preparations were deposited on conventional Balzer’s carrier sample and then rapidly frozen in liquid Freon-22. Fracturing and replication were done using a Balzer’s BAF 301 apparatus in a conventional way. The replicas were cleaned in clorox, rinsed with distilled water, and observed with a Siemens IA electron microscope. The pellets of intact gap junction and insoluble material were processed for electron microscopy as described previously [4].
RESULTS After incubation of the gap junctions in the detergent mixture of digitonin and octyl glucoside, the insoluble 312
SHORT
313
NOTE
fracture plane goes through adjacent membranes, the pits on the upper membrane closely correspond to the particles within the lower membrane (Fig. 2D). DISCUSSION
FIG. 1. Electrophoretic profile of gap junctions at different stages of the experiment. The protein composition of the insoluble material after detergent action (lane A) is similar to that of isolated gap junctions (lane C). The protein composition of the solubilized fraction was assayed after reconstitution into liposomes (lane B). The band profile of lane B is similar to that of lanes A and C and exhibits the same major band at 28 kDa. Markers: phosphorylase B, 92.5 kDa; bovine serum albumine, 66.2 kDa; ovalbumine, 45 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor: 21.5 kDa; and lysozyme: 14.4 kDa.
part of the initial material was centrifugated (1 h, 110,OOOg).The pellet was amorphous, indicating that the native structure of gap junctions had been completely destroyed [4]. We compare the protein compositions of the pellet and of the supernatant with the intact isolated liver gap junctions (Fig. 1). The supernatant was reconstituted into liposomes before SDS-PAGE analysis. The presence of proteins in the reconstituted sample (Fig. 1, lane B) proves that proteins have been solubilized by the detergent mixture although the solubilization was incomplete as indicated by the content of the pellet sample (Fig. 1, lane A). At all the stages of the experiment, the band pattern exhibits the major 27- to 2%kDa protein constituent of the liver gap junctions and a less important band of higher molecular weight. The similar electrophoresis profile of soluble and insoluble material to the intact gap junctions suggests that the detergent action is not selective. The solubilized fraction was reconstituted into liposomes at two different protein-to-lipid ratios. At low protein-to-lipid ratio we found the liposomes to be unilamellar vesicles with a small average diameter of 57 nm on freeze-fracture replicas. On the fracture faces of isolated liposomes, clusters of particles were often observed (Fig. 2B). When the vesicles were aggregated into pairs, a few particles appeared localized in the contact area (Fig. 2A). At high protein-to-lipid ratio (Figs. 2C and 2D) the density of particles within the fractures faces is greater and the reconstituted material may contain various types of vesicles in a single preparation. Aggregated particles in disordered arrays are visible on multilamellar liposomes (Fig. 2C). Large unilamellar vesicles carry randomlv dispersed particles of 7 to 12 nm. When the
The present results show that aggregates of particles are present in the vesicles reconstituted from solubilized liver gap junctions. The high-speed centrifugation (1 h, 110,OOOg)step ensures that these aggregates were absent from the starting material for reconstitution but were formed either at the time of the lipid addition or during the process of detergent removal. Two characteristics of the particle aggregates may be compared to the intact gap junctions. The association of two connexons from adjacent membranes seems to be recovered. This may be attributed both to a nondenaturing action of the detergents used and to the detergent removal by dialysis, a smooth method for reconstituting a lipid bilayer around solubilized membrane proteins. The aggregates formed in the plane of the membrane are reminiscent of in situ gap junctions. The requirement of digitonin to disrupt the gap junction structure suggests that cholesterol is involved in the junction assembly. This is consistent with the finding that the gap junction assembly between reaggregating Novikoff hepatoma cells depends on the cholesterol content of the culture medium [12]. The question arises why aggregates can be reconstituted in the presence of digitonin since, at every protein-to-lipid ratio used here, the added cholesterol should be chelated by the excess of digitonin. Assuming that the affinity of the cholesterol for gap junctions is of the same order of magnitude as that of the cholesterol-digitonin complex, one could explain both the incomplete solubilization and the possibility of reconstitution of particle assemblies. The protein composition of the reconstituted liposomes is identical to that of the intact isolated gap junctions. Characteristically, in both cases, the major proteins migrate at 27-28 kDa and a higher weight band is present. This suggests that the action of the detergents is not selective and that all the subunits present in the intact isolated gap junctions are spontaneously reincorporated into a gap junction-like structure. Aside from the restoration of the permeability and electrical activity properties of gap junctions, the characteristic assembly of particles is the best assay for functional preservation. In vitro channel activity has already been reported for the 27- to 2%kDa subunit solubilized from liver with SDS [13]. But without checking the structure restoration it is not easy to evaluate the degree of reconstitution. The present report is considered a first and necessary step toward the full reconstitution of gap junction activity.
314
SHORT
NOTE
FIG. 2. Solubilized fraction reconstituted into liposomes at two different protein concentrations. At low protein-to-lipid ratio, clusters of few particles are visible in the contact area between two vesicles (A: arrow) and on one side of single vesicles (B). Bar: 43.5 nm. At high proteinto-lipid ratio, the reconstituted material contains several kinds of vesicles. In multilamellar liposomes (C), the particles tend to aggregate into disordered arrays (bar: 128 nm). On another micrograph of the same preparation (D), the single arrow indicates a vesicle with a piece of adherent membrane where pits correspond with the particles of the vesicle membrane. The double arrows indicates the presence of pits and particles on the fracture face of a single vesicle. Bar: 109 nm.
We thank Dr. E. L. Benedetti and Dr. I. Dunia for providing helpful facilities to work in their laboratory (Inst. J. Monod, Univ. Paris VII, Paris) and helpful discussion and M. Recouvreur for his technical assistance. REFERENCES 1.
Henderson, 193-218.
D., Eibl, H., and Weber, K. (1979) J. Mol. Bid. 132,
2. 3.
4. 5.
Hertzberg, E. L., and Gilula, N. B. (1979) J. Biol. Chem. 264, 2138-2147. Nicholson, B. J., Hukapiller, M. W., Grim, L. B., Hood, L. L., and Revel, J.-P. (1981) Proc. N&l. Acad. Sci. USA 78, 75947598. Mazet, F., and Blattmann, A. (1988) C. R. Acad. Sci. Paris 307, 679-684. Gooden, M., Rintoul, D., Takekana, M., and Takemoto, L. (1985) Biochem. Biophys. Res. Commun. 128,993-999.
SHORT 6.
10.
Claassen, D. E., and Spooner, B. S. (1988) Bit&em. Biophys. Res.
M. (1985) Proc. N&l.
11.
Sci. USA 82,846~~8472. C. (1985) J. Memb. Bid. 83, 217-
12.
Mazet, F., and Mazet, J.-L. (1989) Molecular and Cell Biology of Gap Junctions, July 18-23, Irsee, FRG. Meyer, R., Malewicz, B., Baumann, W., and Johnson, R. G. (1987) J. CellBiol. 105,228a.
Nikaido,
H., and Rosenberg, E. Y. (1985) J. Membr. Bid. 86,87-
92. 7.
Commun. 154,194-198.
Zampighi,
Ad. 8.
G. A., Hall, J. E., and Kreman,
Girsch, S. J., and Peracchia,
225. 9.
315
NOTE
Dunia, I., Manenti,
S., Rousselet, A., and Benedetti,
J. Cell Biol. 105,1679-1689. Received October 2,1989
E. L. (1987)
13.
Young, J. D. E., Cohn, D. A., and Gilula,
733-743.
N. B. (1987) Cell 48,