Chapter
21
Isolation of Nzcclear Proteins Associated with the Nzcclear Pore Complex and the Nzcclear Peripheral Lamina of Rat Liver ROBERT PETER AARONSON Department of Microbiology. Mt. Sinai School of Medicine, New York, New York
I. Introduction Nuclear pore complexes are ubiquitous ( 1 ) morphologically well-characterized (2,3)organelles situated in pores ( 4 ) characteristic of the bileaflet membrane surrounding the cell nucleus. The nuclear peripheral lamina ( 5 ) has been described less often and occurs in a variety of forms (5-9) as a layer of relatively constant thickness subsuming the inner nuclear membrane. Although the presence of a nuclear peripheral lamina has not been reported in rat liver nuclei, a subnuclear fraction exhibiting laminas with characteristics consistent with a nuclear peripheral lamina can be isolated from rat liver (10). The laminas extend for several microns, do not have a unit membrane structure, and exhibit nuclear pore complexes attached in a specific orientation. This subnuclear fraction is at least 95% protein, and is made up of approximately equal amounts of three polypeptide species as shown upon analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The protein of the nuclear pore complex-peripheral lamina fraction represents approximately 5% of the total protein of rat liver nuclei. Isolation of the subnuclear fraction containing proteins associated with the nuclear pore complexes and the peripheral lamina is achieved in three steps: isolation of pure nuclei by an aqueous procedure (11), preparation of intact nuclear envelopes by deoxyribonuclease digestion of chromatin under alkaline conditions (12), and solubilization of lipid (13) and contaminating chromatin. The original procedure (10)relied on the use of a high concentra337
338
ROBERT PETER AARONSON
tion of MgCl, to solubilizethe remaining chromatin. High concentrations of NaCl are equally effective (G. Blobel, personal communication). An alternate procedure is also presented utilizing the polyanion polyvinylsulfate during DNase digestion. Inclusion of the polyvinyl sulfate allows complete hydrolysis of the DNA, thus permitting isolation of the nuclear pore complexfibrous lamina subfraction without subjecting it to high salt concentrations.
11. Method A. Isolation of Rat Liver Nuclei (22) Four 150-gm male rats are fasted overnight (approximately 18hours) and sacrificed by decapitation using a Harvard guillotine. The livers are removed, rinsed briefly with ice-cold deionized water, and then minced in ice-cold 0.25 M sucrose-50 mM Tris-C1 (pH 7.5)-25 mM KC1-5 mM MgCl, (0.25s-TKM). All further steps in isolating nuclei are performed at 4" or in ice-cold buffer. The pieces of liver are rinsed twice and then homogenized with approximately 2 volumes of 0.25s-TKM in 40-ml batches with five up-and-down strokes of a loose-fitting Teflon-pestle Potter-Elvehjem homogenizer. The homogenate is filtered through a nylon monofilament screen with 80-pm openings (Nitex HC3-100, Tetko, Inc., Elmsford, New York). Then 80 ml of the homogenate are mixed well with 160 ml of 2.3 M sucrose-50 mM Tris-C1(pH7.5)-25mMKC1-5 mMMgC1, (2.3s-TKM). The mixture is distributed equally to six cellulose nitrate tubes (35 ml per tube) which fit the Beckman SW27 rotor. Then 5 ml of 2.3s-TKM are added at the top of each tube. As the 2.3s-TKM settles to the bottom of the tube a small amount of mixing with homogenate occurs so that the resulting cushion does not have a sharp boundary. The tubes are then centrifuged for 1 hour at 25,000 rpm at 40. The nuclei form a white pellet. The supernatant is poured off along with the congealed layer of red cells, cell debris, and mitochondria at the top of the tubes. The tube walls are wiped clean and the pellet of nuclei is gently disrupted with a spatula and resuspended in a small volume of 2.3s-TKM using a Vortex mixer. The suspension of nuclei is diluted to 40 ml with 0.25s-TKM and centrifuged at 1000 rpm for 20 minutes at 4 in a Beckman JS-13 rotor (gav = 100). The nuclei are resuspended in 40 ml of0.25S-TKMY and a 20 p1 aliquot is diluted to 1 ml with 0.25s-TKM. The absorbance of the dilute nuclear suspension at 260 nm is approximately 0.25 OD. The nuclear suspension is poured slowly into another tube, leaving small clumps of aggregated nuclei behind, and then centrifuged at lo00 rpm for 20 minutes as above. The supernatant is removed by aspiration leaving a
21.
NUCLEAR PORE COMPLEX A N D NUCLEAR PERIPHERAL LAMINA
339
loose white pellet of approximately 500 A,,, units of nuclei. There are 3.0 k 0.3 x lo6 nuclei per A,,, unit.
B. Preparation of Nuclear Envelopes from Isolated Rat Liver Nuclei (12) To the loose pellet of nuclei add, slowly initially with gentle mixing, 125ml of 0.25 M sucrose-I0 mM Tris-C1 (PH 8.5)a.l mM MgC1,-1 pglml bovine pancreatic DNase I at 23 ". Swirl gently at 5-minute intervals for 15 minutes at 23" before adding 125 ml of ice-cold deionized water. Centrifuge the partially digested suspension of nuclei at 3000 rpm for 10 minutes at 40in the Beckman JS-13 rotor (gay= 900). Remove the supernatant by aspiration. Gently resuspend the pellet with 125 ml of 0.25 M sucrose-10 mM Tris-C1 (pH 7 . 5 ) a . l mM MgC1,-1 ,tg/ml DNase I at 23". Swirl gently at 5minute intervals for 20 minutes before adding 125 ml of ice-cold deionized water. Centrifuge at 3000 rpm for 15 minutes at 4 " as above. The supernatant is removed by aspiration leaving a small white pellet of nuclear envelopes.
C . Solubilization of Lipid and Chromatin (13,24) 1. The pellet of nuclear envelopes is carefully suspended with 2 ml of icecold 0.25s-TKM. To this add slowly 0.5 ml of ice-cold 10%Triton X-100 and let stand at 0" for 10 minutes. Centrifuge at 3000 rpm for 15 minutes at 4 " as above. Remove the supernatant by aspiration leaving the small pellet of phospholipid-depleted material. 2. The pellet is suspended with 5 ml of ice-cold 0.25s-TKM. Add 2.5 ml of cold 1 M MgCl, or 5 ml of cold 2 M NaCI. Centrifuge at 11,000 rpm for 20 minutes at 4 " in the JS-13 rotor (gay= 12,000). The pellet consisting of the nuclear pore complex-peripheral lamina contains up to 3 mg of protein.
D. Alternate Procedure for the Preparation of Nuclear Envelopes from Isolated Rat Liver Nuclei and DetergentSolubilization of Phospholipid 1. To the loose pellet of nuclei add, slowly initially with gentle mixing, 125 ml of 10 mM Tris-Cl (pH 8.5)a.l mM MgCl,-O.l mM CaC1,-1 pg/ml bovine pancreatic DNase I at 23 Immediately add 2.5 ml of 10 mg/ml polyvinylsulfate. Swirl at 5-minute intervals for 30 minutes. Centrifuge at 3000 rpm for 30 minutes at 4" in the Beckman JS-13 rotor (gay= 900). Suspend the pellet with 30 ml of 10 mM Tris-C1 (pH 8 . 5 ) a . l mM MgCl,-O.l mM CaC1, and centrifuge as above. Remove the supernatant by aspiration leaving a pellet of nuclear envelopes. O.
340
ROBERT PETER AARONSON
FIG. 1. The subnuclear fraction containing nuclear pore complexes in association with the nuclear peripheral lamina. This survey micrograph demonstrates the extensiveness and flexibility of the lamina. The profiles observed in these thin sections are consistent with the possibility that the nuclear peripheral lamina subsumes large areas, if not all, of the inner nuclear membrane. x l0,OOO. The bar indicates 1 pm. RG.2. A higher magnification of a portion of Fig. 1 demonstrating in enfuce and lateral views the specific orientation and attachment of figures resembling the nuclear pore complex to a folded portion of the nuclear peripheral lamina. x 61,000. The bar indicates 100 nm.
21.
341
NUCLEAR PORE COMPLEX AND NUCLEAR PERIPHERAL LAMINA
2. Suspend the nuclear envelope pellet in 2 ml of ice-cold 10 mMTrisC1 (PH 8.5)a.l mM MgC124.1 mM CaCl, and add 0.5 ml of ice-cold 10% Triton X-100. Incubate at 0 0 for 10 minutes and centrifuge at 11,000rpm for 20 minutes at 4" in the Beckman JS-13 rotor (gav= 12,000). Remove the supernatant by aspiration. The pellet contains the nuclear pore complexperipheral lamina.
111. Comments The nuclear pore complex-peripheral lamina is a delicate structure whose integrity depends on the lack of agitation during preparation and gentle sedimentation. Optimal morphological preservation necessitates fixation for electron microscopy in suspension prior to the final centrifugation. Figures 1 and 2 demonstrate the appearance of a preparation of nuclear pore complex-fibrous lamina which was fixed in suspension overnight at 4" in 1% glutaraldehyde-0.1 M triethanolamine-C1 (pH 8.5) prior to centrifugation. Both circular figures and goblet-shaped figures resembling nuclear pore complexes are arrayed, with a specific orientation, on a rather extensive flexible 15-nm thick amorphous lamina. Figure 3 demonstrates that the pore complex-peripheral lamina subfraction consists primarily of three polypeptides with similar molecular weights. These three polypeptides (or classes of polypeptides) appear to be present in nearly equivalent amounts. Furthermore, it can be seen that more common structural proteins [e.g., tubulin (52,000 MW), actin (44,000 M W ) , and myosin (190,000 MW)] are, if present, negligible components of the subfraction. 17
23
103
~103
I
I
45 103
I
67 103
I
135
190
x ~ 0 3 x ~ 0 3
I
I
d
FIG. 3. Densitometer recording of SDS-PAGE analysis of the nuclear pore complexperipheral lamina subfraction. The subfraction was solubilized in sodium dodecyl sulfate, heated under reducing conditions at 100" for 3 minutes, alkylated with iodoacetamide, and subjected to electrophoresis in the presence of sodium dodecyl sulfate on a 150 mm x I mm slab gradient (10-1 5% polyacrylamide) gel. The short vertical lines indicate the positions of migration of protein standards (molecular weights noted) applied in an adjacent slot.
342
ROBERT PETER AARONSON
ACKNOWLEDGMENT This research was supported in part by grant GM 21950 from the National Institutes of Health.
REFERENCES 1. Franke, W. W., Z . Zelrforsch. Mikrosk. Anat. 105,405, 1970. 2. Feldherr, C., A h . Cell Molec. Biol. 2,273 (1972). 3. Kessel, R. G. Progr. Surface Membrane Sci. 6 , 243 (1973). 4 . Stevens, B. J., and Andrt, J., in “Handbook of Molecular Cytology” (A. Lima-de-Faria, ed.),p. 837. North-Holland h b l . Co., Amsterdam, 1969. 5. Fawcett, D. W., Am. J. Anat. 119, 129 (1966). 6. Gall, J. G., in “Protoplasmatologia, Handbuch der Protoplasmaforschung.” Vol. 5, p. 4. Springer-Verlag, Berlin and New York, 1964. 7. Patrizi, G., and Poger, M., J. Ultrastruct. Res. 17, 127 (1967). 8. Kalifat, S. R., Bouteille, M., and Delarue, J., J. Microscopie6, 1019 (1967). 9. Mazanec, K., J. Microsc. (Paris) 6 , 1027 (1967). 10. Aaronson. R. P., and Blobel, G., Proc. Natl. Acad. Sci. U S A . 72, 1007 (1975). 11. Blobel, G., and Potter, V. R., Science 154, 1662 (1966). 12. Kay, R. R., Fraser, D., and Johnston, I. R., Eur. J. Biochem. 30, 145 (1972). 13. Aaronson, R. P., and Blobel, G., J. CeNBiol. 62, 746 (1973). 14. Monneron, A., Blobel, G., and Palade, G. E., J. CellBiol. 55, 104 (1972).
21
Isolation of Nzcclear Proteins Associated with the Nzcclear Pore Complex and the Nzcclear Peripheral Lamina of Rat Liver ROBERT PETER AARONSON Department of Microbiology. Mt. Sinai School of Medicine, New York, New York
I. Introduction Nuclear pore complexes are ubiquitous ( 1 ) morphologically well-characterized (2,3)organelles situated in pores ( 4 ) characteristic of the bileaflet membrane surrounding the cell nucleus. The nuclear peripheral lamina ( 5 ) has been described less often and occurs in a variety of forms (5-9) as a layer of relatively constant thickness subsuming the inner nuclear membrane. Although the presence of a nuclear peripheral lamina has not been reported in rat liver nuclei, a subnuclear fraction exhibiting laminas with characteristics consistent with a nuclear peripheral lamina can be isolated from rat liver (10). The laminas extend for several microns, do not have a unit membrane structure, and exhibit nuclear pore complexes attached in a specific orientation. This subnuclear fraction is at least 95% protein, and is made up of approximately equal amounts of three polypeptide species as shown upon analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The protein of the nuclear pore complex-peripheral lamina fraction represents approximately 5% of the total protein of rat liver nuclei. Isolation of the subnuclear fraction containing proteins associated with the nuclear pore complexes and the peripheral lamina is achieved in three steps: isolation of pure nuclei by an aqueous procedure (11), preparation of intact nuclear envelopes by deoxyribonuclease digestion of chromatin under alkaline conditions (12), and solubilization of lipid (13) and contaminating chromatin. The original procedure (10)relied on the use of a high concentra337
338
ROBERT PETER AARONSON
tion of MgCl, to solubilizethe remaining chromatin. High concentrations of NaCl are equally effective (G. Blobel, personal communication). An alternate procedure is also presented utilizing the polyanion polyvinylsulfate during DNase digestion. Inclusion of the polyvinyl sulfate allows complete hydrolysis of the DNA, thus permitting isolation of the nuclear pore complexfibrous lamina subfraction without subjecting it to high salt concentrations.
11. Method A. Isolation of Rat Liver Nuclei (22) Four 150-gm male rats are fasted overnight (approximately 18hours) and sacrificed by decapitation using a Harvard guillotine. The livers are removed, rinsed briefly with ice-cold deionized water, and then minced in ice-cold 0.25 M sucrose-50 mM Tris-C1 (pH 7.5)-25 mM KC1-5 mM MgCl, (0.25s-TKM). All further steps in isolating nuclei are performed at 4" or in ice-cold buffer. The pieces of liver are rinsed twice and then homogenized with approximately 2 volumes of 0.25s-TKM in 40-ml batches with five up-and-down strokes of a loose-fitting Teflon-pestle Potter-Elvehjem homogenizer. The homogenate is filtered through a nylon monofilament screen with 80-pm openings (Nitex HC3-100, Tetko, Inc., Elmsford, New York). Then 80 ml of the homogenate are mixed well with 160 ml of 2.3 M sucrose-50 mM Tris-C1(pH7.5)-25mMKC1-5 mMMgC1, (2.3s-TKM). The mixture is distributed equally to six cellulose nitrate tubes (35 ml per tube) which fit the Beckman SW27 rotor. Then 5 ml of 2.3s-TKM are added at the top of each tube. As the 2.3s-TKM settles to the bottom of the tube a small amount of mixing with homogenate occurs so that the resulting cushion does not have a sharp boundary. The tubes are then centrifuged for 1 hour at 25,000 rpm at 40. The nuclei form a white pellet. The supernatant is poured off along with the congealed layer of red cells, cell debris, and mitochondria at the top of the tubes. The tube walls are wiped clean and the pellet of nuclei is gently disrupted with a spatula and resuspended in a small volume of 2.3s-TKM using a Vortex mixer. The suspension of nuclei is diluted to 40 ml with 0.25s-TKM and centrifuged at 1000 rpm for 20 minutes at 4 in a Beckman JS-13 rotor (gav = 100). The nuclei are resuspended in 40 ml of0.25S-TKMY and a 20 p1 aliquot is diluted to 1 ml with 0.25s-TKM. The absorbance of the dilute nuclear suspension at 260 nm is approximately 0.25 OD. The nuclear suspension is poured slowly into another tube, leaving small clumps of aggregated nuclei behind, and then centrifuged at lo00 rpm for 20 minutes as above. The supernatant is removed by aspiration leaving a
21.
NUCLEAR PORE COMPLEX A N D NUCLEAR PERIPHERAL LAMINA
339
loose white pellet of approximately 500 A,,, units of nuclei. There are 3.0 k 0.3 x lo6 nuclei per A,,, unit.
B. Preparation of Nuclear Envelopes from Isolated Rat Liver Nuclei (12) To the loose pellet of nuclei add, slowly initially with gentle mixing, 125ml of 0.25 M sucrose-I0 mM Tris-C1 (PH 8.5)a.l mM MgC1,-1 pglml bovine pancreatic DNase I at 23 ". Swirl gently at 5-minute intervals for 15 minutes at 23" before adding 125 ml of ice-cold deionized water. Centrifuge the partially digested suspension of nuclei at 3000 rpm for 10 minutes at 40in the Beckman JS-13 rotor (gay= 900). Remove the supernatant by aspiration. Gently resuspend the pellet with 125 ml of 0.25 M sucrose-10 mM Tris-C1 (pH 7 . 5 ) a . l mM MgC1,-1 ,tg/ml DNase I at 23". Swirl gently at 5minute intervals for 20 minutes before adding 125 ml of ice-cold deionized water. Centrifuge at 3000 rpm for 15 minutes at 4 " as above. The supernatant is removed by aspiration leaving a small white pellet of nuclear envelopes.
C . Solubilization of Lipid and Chromatin (13,24) 1. The pellet of nuclear envelopes is carefully suspended with 2 ml of icecold 0.25s-TKM. To this add slowly 0.5 ml of ice-cold 10%Triton X-100 and let stand at 0" for 10 minutes. Centrifuge at 3000 rpm for 15 minutes at 4 " as above. Remove the supernatant by aspiration leaving the small pellet of phospholipid-depleted material. 2. The pellet is suspended with 5 ml of ice-cold 0.25s-TKM. Add 2.5 ml of cold 1 M MgCl, or 5 ml of cold 2 M NaCI. Centrifuge at 11,000 rpm for 20 minutes at 4 " in the JS-13 rotor (gay= 12,000). The pellet consisting of the nuclear pore complex-peripheral lamina contains up to 3 mg of protein.
D. Alternate Procedure for the Preparation of Nuclear Envelopes from Isolated Rat Liver Nuclei and DetergentSolubilization of Phospholipid 1. To the loose pellet of nuclei add, slowly initially with gentle mixing, 125 ml of 10 mM Tris-Cl (pH 8.5)a.l mM MgCl,-O.l mM CaC1,-1 pg/ml bovine pancreatic DNase I at 23 Immediately add 2.5 ml of 10 mg/ml polyvinylsulfate. Swirl at 5-minute intervals for 30 minutes. Centrifuge at 3000 rpm for 30 minutes at 4" in the Beckman JS-13 rotor (gay= 900). Suspend the pellet with 30 ml of 10 mM Tris-C1 (pH 8 . 5 ) a . l mM MgCl,-O.l mM CaC1, and centrifuge as above. Remove the supernatant by aspiration leaving a pellet of nuclear envelopes. O.
340
ROBERT PETER AARONSON
FIG. 1. The subnuclear fraction containing nuclear pore complexes in association with the nuclear peripheral lamina. This survey micrograph demonstrates the extensiveness and flexibility of the lamina. The profiles observed in these thin sections are consistent with the possibility that the nuclear peripheral lamina subsumes large areas, if not all, of the inner nuclear membrane. x l0,OOO. The bar indicates 1 pm. RG.2. A higher magnification of a portion of Fig. 1 demonstrating in enfuce and lateral views the specific orientation and attachment of figures resembling the nuclear pore complex to a folded portion of the nuclear peripheral lamina. x 61,000. The bar indicates 100 nm.
21.
341
NUCLEAR PORE COMPLEX AND NUCLEAR PERIPHERAL LAMINA
2. Suspend the nuclear envelope pellet in 2 ml of ice-cold 10 mMTrisC1 (PH 8.5)a.l mM MgC124.1 mM CaCl, and add 0.5 ml of ice-cold 10% Triton X-100. Incubate at 0 0 for 10 minutes and centrifuge at 11,000rpm for 20 minutes at 4" in the Beckman JS-13 rotor (gav= 12,000). Remove the supernatant by aspiration. The pellet contains the nuclear pore complexperipheral lamina.
111. Comments The nuclear pore complex-peripheral lamina is a delicate structure whose integrity depends on the lack of agitation during preparation and gentle sedimentation. Optimal morphological preservation necessitates fixation for electron microscopy in suspension prior to the final centrifugation. Figures 1 and 2 demonstrate the appearance of a preparation of nuclear pore complex-fibrous lamina which was fixed in suspension overnight at 4" in 1% glutaraldehyde-0.1 M triethanolamine-C1 (pH 8.5) prior to centrifugation. Both circular figures and goblet-shaped figures resembling nuclear pore complexes are arrayed, with a specific orientation, on a rather extensive flexible 15-nm thick amorphous lamina. Figure 3 demonstrates that the pore complex-peripheral lamina subfraction consists primarily of three polypeptides with similar molecular weights. These three polypeptides (or classes of polypeptides) appear to be present in nearly equivalent amounts. Furthermore, it can be seen that more common structural proteins [e.g., tubulin (52,000 MW), actin (44,000 M W ) , and myosin (190,000 MW)] are, if present, negligible components of the subfraction. 17
23
103
~103
I
I
45 103
I
67 103
I
135
190
x ~ 0 3 x ~ 0 3
I
I
d
FIG. 3. Densitometer recording of SDS-PAGE analysis of the nuclear pore complexperipheral lamina subfraction. The subfraction was solubilized in sodium dodecyl sulfate, heated under reducing conditions at 100" for 3 minutes, alkylated with iodoacetamide, and subjected to electrophoresis in the presence of sodium dodecyl sulfate on a 150 mm x I mm slab gradient (10-1 5% polyacrylamide) gel. The short vertical lines indicate the positions of migration of protein standards (molecular weights noted) applied in an adjacent slot.
342
ROBERT PETER AARONSON
ACKNOWLEDGMENT This research was supported in part by grant GM 21950 from the National Institutes of Health.
REFERENCES 1. Franke, W. W., Z . Zelrforsch. Mikrosk. Anat. 105,405, 1970. 2. Feldherr, C., A h . Cell Molec. Biol. 2,273 (1972). 3. Kessel, R. G. Progr. Surface Membrane Sci. 6 , 243 (1973). 4 . Stevens, B. J., and Andrt, J., in “Handbook of Molecular Cytology” (A. Lima-de-Faria, ed.),p. 837. North-Holland h b l . Co., Amsterdam, 1969. 5. Fawcett, D. W., Am. J. Anat. 119, 129 (1966). 6. Gall, J. G., in “Protoplasmatologia, Handbuch der Protoplasmaforschung.” Vol. 5, p. 4. Springer-Verlag, Berlin and New York, 1964. 7. Patrizi, G., and Poger, M., J. Ultrastruct. Res. 17, 127 (1967). 8. Kalifat, S. R., Bouteille, M., and Delarue, J., J. Microscopie6, 1019 (1967). 9. Mazanec, K., J. Microsc. (Paris) 6 , 1027 (1967). 10. Aaronson. R. P., and Blobel, G., Proc. Natl. Acad. Sci. U S A . 72, 1007 (1975). 11. Blobel, G., and Potter, V. R., Science 154, 1662 (1966). 12. Kay, R. R., Fraser, D., and Johnston, I. R., Eur. J. Biochem. 30, 145 (1972). 13. Aaronson, R. P., and Blobel, G., J. CeNBiol. 62, 746 (1973). 14. Monneron, A., Blobel, G., and Palade, G. E., J. CellBiol. 55, 104 (1972).
