Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5400-5404, November 1978
Biochemistry
Activation energy for RNA transport from isolated rat liver nuclei (Arrhenius graph/inward diffusion/RNase activity)
GARY A. CLAWSON AND EDWARD A. SMUCKLER* Department of Pathology, University of California School of Medicine, San Francisco, California 94143
Communicated by Earl P. Benditt, August 24, 1978
ABSTRACr The temperature dependence of ATP-enhanced RNA delivery from rat liver nuclei to a surrogate cytoplasm was investigated. Examination of linear-rate data on Arrhenius graphs of l/T vs. log (% RNA delivered per min) revealed an activation energy of 12.5-13 kcal/mol. When data derived from longer incubation periods was displayed on Arrhenius graphs, we observed a discontinuous graph-two distinct linear segments with slopes of differing sign which intersected near 20°C. It was demonstrated that this discontinuity was not due to lipid phase transition in the nuclear membranes and that its position depended upon treatment of the nuclei and upon additives to the incubation mixtures. The decline in transport apparent in the upper-temperature domain on 20-min Arrhenius graphs was shown to be based on the diffusion of transported macromolecular RNA back into the nucleus-a process greatly amplified by the rapidity of transport in this domain. The large net inward diffusion, in concert with significantly differing activation energies for RNA transport and passive diffusion, suggests that the process of nucleocytoplasmic RNA transport is not diffusion driven. Our data have established that an integral parameter of RNA transport (namely, the activation energy) remains unchanged in various in vitro manipulations.
Numerous in vitro assay systems for the nuclear transport of RNA have been reported. Schneider's (1) model demonstrated the effects of nucleotides on the rate of RNA release. Many other investigators (2-12) have employed variant systems to define the effects of added factors-e.g., cell-sap components (7, 9), chelating agents (10, 12), detergents (7), and metabolic inhibitors (1, 12)-on both the rate of RNA transport and the properties of the released material (2, 3). Although the influence of added nucleoside triphosphates upon the RNA transport rate has been confirmed (2-5, 8), the wide range of relative rates obtained under diverse incubation conditions complicates correlation of this reported data and the transport process itself. In this communication, we present evidence that the activation energy for transporting trichloroacetic acid-precipitable RNA from the rat liver nucleus to the incubation environment remains unaltered despite considerable modification of the rate of RNA transport by treatments and additions at particular temperatures.
MATERIALS AND METHODS Male pathogen-free Sprague-Dawley rats were obtained from the Charles River Breeding Laboratories and maintained in our vivarium for at least 5 days before experimentation. RNA Labeling. The animals were starved overnight (15 hr) prior to radioactive labeling. Under light ether anesthesia, they were given single intravenous injections of ['4C]orotic acid (New England Nuclear), 28 mCi/mmol at 3 ,Ci/100 g of body weight, via the tail vein, and killed 45 min later. Isolation of Nuclei. The livers were homogenized in Teflon/glass homogenizers with sucrose buffer (0.25 M RNase-free The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
sucrose/5 mM mercaptoethanol/50 mM Tris-HCl, pH 7.4/25 mM KCI/5 mM MgCl2) at 00, and mixed with 2.3 M RNasefree sucrose buffer with the same composition (e.g., KCl, MgCl2, mercaptoethanol). This suspension was laid over a "cushion" of 2.3 M sucrose buffer and centrifuged in an SW27 rotor at 95,000 X g for 75 min at SOC (13). Nuclei were recovered after passage through the 2.3 M sucrose buffer, resuspension in 0.88 M RNase-free sucrose (pH 7.6) containing 50 mM Tris-HCl, 5 mM MgCl2, and 25 mM KC1, and dilution to a protein concentration of 7.4 mg/ml. To isolate detergent-treated nuclei, we added a solution of Triton X-100 (Packard Instruments) and Nonidet P-40 (Shell Oil) (final concentrations, both 0.5%) to the nuclear suspension; the material was mixed thoroughly with a Vortex mixer. The nuclei were resedimented at 600 X g for 10 min at 0C. The nuclei were again resuspended to the original volume in 0.88 M sucrose buffer. Preparation of Cell Sap. Cell sap was prepared as the 105,000 X g-supernatant fraction of the liver homogenate which was dialyzed against 0.88 M sucrose buffer overnight before use. Cell sap preparations were used fresh at final concentrations of 8-10 mg of protein per ml. RNA Release Assay. Nuclei at a final protein concentration of 3.7 mg/ml were incubated with 0.88 M sucrose buffer and purified RNase inhibitor or other additions (see Table 2-final nucleotide concentration at 7.5 mM unless otherwise indicated) as described (4). Nuclei were incubated in the different mixtures for 20 min at various temperatures (every 5°C-interval over the 35-0°C domain). The reactions were terminated by the addition of 4 vol of ice-cold 0.88 M sucrose buffer, and the nuclei were sedimented by centrifugation at 800 X g for 8 min. Additionally, in the upper-temperature domain (35-20'C), incubations were conducted for 5-, 10-, 20-, and 30-min periods. For assaying released radioactivity, this supernatant was withdrawn and precipitated with trichloroacetic acid at a final concentration of 10%. The precipitated material was recovered by centrifugation at 1000 X g for 30 min and the pellet was dissolved in 5 M NaOH; radioactivity was measured in a liquid scintillation counter. Supernatant radioactivity at 0 time was subtracted to obtain the amount released. For determination of nuclear acid-precipitable radioactivity (used as a percentage standard), the nuclei were resuspended and lysed in 1.0% sodium dodecyl sulfate, and the mixture was acid-precipitated on ice. The precipitate was collected on Whatman GF/C filters, dried, and counted in liquid scintillant; recovery under these conditions was 40% of total radioactivity. Total acid-precipitable radioactivity (nuclear plus supernatant fractions) was monitored. With RNase inhibitor present, the total acid-precipitable radioactivity remained essentially constant for 30 min. Assay for Nuclear Uptake of RNA. Nuclei were labeled and isolated as described above and incubated for 10 min at various * To whom correspondence and reprint requests should be addressed.
5400
Biochemistry: Clawson and Smuckler
Proc. Natl. Acad. Sci. USA 75 (1978)
(as indicated in tables). After incubation, the nuclei were removed by centrifugation; the supernatant ("labeled supernatant") was withdrawn and frozen at -70'C. Unlabeled nuclei were isolated, incubated in similar fashion, recovered by centrifugation, resuspended in the "labeled supernatant," and incubated for 0, 5, 10, or 20 min. The reactions were stopped by the addition of 4 vol of ice-cold 0.88 M sucrose buffer, and the supernatant and nuclear acid-precipitable material were collected on Whatman GF/C filters, washed repeatedly, and counted as described. Alternately, Escherichia coli [3H]tRNA (Miles Laboratories, 43,gCi/ml, approximately 50,000 cpm/aliquot) was mixed in appropriate proportion with ATP, RNase inhibitor, and sucrose buffer; and unlabeled nuclei were resuspended in this incubation mixture. At 0 time or after incubation for 5, 10, or 20 min, the nuclei were sedimented by centrifugation, and the temperatures
nuclear and supernatant acid-precipitable material was measured as described. Statistical Analysis of Data. Linear regression coefficients were calculated by the method of least squares. Analyses were performed in each individual experiment and the results were grouped. RNA transport data at the various temperatures were homoscedastic. We tested the significance of fit as the F value of the ratio of explained variation to unexplained variation, or the corresponding t value. Analysis of covariance was performed on the calculated regression coefficients, and the significance of differences was tested with F values. RESULTS To investigate the energetics of RNA transport from isolated rat liver nuclei, we examined the temperature dependence of RNA in the temperature domain 35-00C. By following our standard protocol, we assessed transport after various incubation periods ranging from 5 to 20 min. When initial linear rates (up to 5 min) of ATP-stimulated transport were measured and used for Arrhenius analysis, a straight line was obtained and the activation energy for the RNA transport process was found to be 13 kcal/mol (1 cal = 4.184 J) (Fig. 1 and Table 1). However, when ATP-dependent transport obtained after a 20-min incubation was exhibited on an Arrhenius plot in the domain up to 370C, we observed two linear segments (one of positive slope and the other of negative slope) intersecting near 20'C. A significant decline in RNA transport (t20% of nuclear standard) was noted at 350C compared to that at 200C, resulting in a "peaked" graph reflecting maximal transport near 20'C. When 20-min transport data from the lower-temperature domain (20-0OC) were utilized in calculations (see Table 2), an activation energy similar to that derived from initial linear rates over the entire temperature domain was obtained, because RNA transport proceeded linearly in this domain (see
below). To ascertain the basis for the progressive decline
in
per-
centage of RNA transported in the upper-temperature domain (35-20° C) in the presence of ATP, we studied the time course
of RNA
transport. Transport was not linear over the 20-min incubation period in this domain; in general, the higher the temperature, the more rapidly maximal transport was observed. At 35'C, for example, 60.5, 57.5, 43.2, and 27% of the'acid-
precipitable nuclear RNA was present in the supernatant after 5-, 10-, 20-, and 30-min incubations (with ATP), respectively. These points were colinear (P
Biochemistry
Activation energy for RNA transport from isolated rat liver nuclei (Arrhenius graph/inward diffusion/RNase activity)
GARY A. CLAWSON AND EDWARD A. SMUCKLER* Department of Pathology, University of California School of Medicine, San Francisco, California 94143
Communicated by Earl P. Benditt, August 24, 1978
ABSTRACr The temperature dependence of ATP-enhanced RNA delivery from rat liver nuclei to a surrogate cytoplasm was investigated. Examination of linear-rate data on Arrhenius graphs of l/T vs. log (% RNA delivered per min) revealed an activation energy of 12.5-13 kcal/mol. When data derived from longer incubation periods was displayed on Arrhenius graphs, we observed a discontinuous graph-two distinct linear segments with slopes of differing sign which intersected near 20°C. It was demonstrated that this discontinuity was not due to lipid phase transition in the nuclear membranes and that its position depended upon treatment of the nuclei and upon additives to the incubation mixtures. The decline in transport apparent in the upper-temperature domain on 20-min Arrhenius graphs was shown to be based on the diffusion of transported macromolecular RNA back into the nucleus-a process greatly amplified by the rapidity of transport in this domain. The large net inward diffusion, in concert with significantly differing activation energies for RNA transport and passive diffusion, suggests that the process of nucleocytoplasmic RNA transport is not diffusion driven. Our data have established that an integral parameter of RNA transport (namely, the activation energy) remains unchanged in various in vitro manipulations.
Numerous in vitro assay systems for the nuclear transport of RNA have been reported. Schneider's (1) model demonstrated the effects of nucleotides on the rate of RNA release. Many other investigators (2-12) have employed variant systems to define the effects of added factors-e.g., cell-sap components (7, 9), chelating agents (10, 12), detergents (7), and metabolic inhibitors (1, 12)-on both the rate of RNA transport and the properties of the released material (2, 3). Although the influence of added nucleoside triphosphates upon the RNA transport rate has been confirmed (2-5, 8), the wide range of relative rates obtained under diverse incubation conditions complicates correlation of this reported data and the transport process itself. In this communication, we present evidence that the activation energy for transporting trichloroacetic acid-precipitable RNA from the rat liver nucleus to the incubation environment remains unaltered despite considerable modification of the rate of RNA transport by treatments and additions at particular temperatures.
MATERIALS AND METHODS Male pathogen-free Sprague-Dawley rats were obtained from the Charles River Breeding Laboratories and maintained in our vivarium for at least 5 days before experimentation. RNA Labeling. The animals were starved overnight (15 hr) prior to radioactive labeling. Under light ether anesthesia, they were given single intravenous injections of ['4C]orotic acid (New England Nuclear), 28 mCi/mmol at 3 ,Ci/100 g of body weight, via the tail vein, and killed 45 min later. Isolation of Nuclei. The livers were homogenized in Teflon/glass homogenizers with sucrose buffer (0.25 M RNase-free The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
sucrose/5 mM mercaptoethanol/50 mM Tris-HCl, pH 7.4/25 mM KCI/5 mM MgCl2) at 00, and mixed with 2.3 M RNasefree sucrose buffer with the same composition (e.g., KCl, MgCl2, mercaptoethanol). This suspension was laid over a "cushion" of 2.3 M sucrose buffer and centrifuged in an SW27 rotor at 95,000 X g for 75 min at SOC (13). Nuclei were recovered after passage through the 2.3 M sucrose buffer, resuspension in 0.88 M RNase-free sucrose (pH 7.6) containing 50 mM Tris-HCl, 5 mM MgCl2, and 25 mM KC1, and dilution to a protein concentration of 7.4 mg/ml. To isolate detergent-treated nuclei, we added a solution of Triton X-100 (Packard Instruments) and Nonidet P-40 (Shell Oil) (final concentrations, both 0.5%) to the nuclear suspension; the material was mixed thoroughly with a Vortex mixer. The nuclei were resedimented at 600 X g for 10 min at 0C. The nuclei were again resuspended to the original volume in 0.88 M sucrose buffer. Preparation of Cell Sap. Cell sap was prepared as the 105,000 X g-supernatant fraction of the liver homogenate which was dialyzed against 0.88 M sucrose buffer overnight before use. Cell sap preparations were used fresh at final concentrations of 8-10 mg of protein per ml. RNA Release Assay. Nuclei at a final protein concentration of 3.7 mg/ml were incubated with 0.88 M sucrose buffer and purified RNase inhibitor or other additions (see Table 2-final nucleotide concentration at 7.5 mM unless otherwise indicated) as described (4). Nuclei were incubated in the different mixtures for 20 min at various temperatures (every 5°C-interval over the 35-0°C domain). The reactions were terminated by the addition of 4 vol of ice-cold 0.88 M sucrose buffer, and the nuclei were sedimented by centrifugation at 800 X g for 8 min. Additionally, in the upper-temperature domain (35-20'C), incubations were conducted for 5-, 10-, 20-, and 30-min periods. For assaying released radioactivity, this supernatant was withdrawn and precipitated with trichloroacetic acid at a final concentration of 10%. The precipitated material was recovered by centrifugation at 1000 X g for 30 min and the pellet was dissolved in 5 M NaOH; radioactivity was measured in a liquid scintillation counter. Supernatant radioactivity at 0 time was subtracted to obtain the amount released. For determination of nuclear acid-precipitable radioactivity (used as a percentage standard), the nuclei were resuspended and lysed in 1.0% sodium dodecyl sulfate, and the mixture was acid-precipitated on ice. The precipitate was collected on Whatman GF/C filters, dried, and counted in liquid scintillant; recovery under these conditions was 40% of total radioactivity. Total acid-precipitable radioactivity (nuclear plus supernatant fractions) was monitored. With RNase inhibitor present, the total acid-precipitable radioactivity remained essentially constant for 30 min. Assay for Nuclear Uptake of RNA. Nuclei were labeled and isolated as described above and incubated for 10 min at various * To whom correspondence and reprint requests should be addressed.
5400
Biochemistry: Clawson and Smuckler
Proc. Natl. Acad. Sci. USA 75 (1978)
(as indicated in tables). After incubation, the nuclei were removed by centrifugation; the supernatant ("labeled supernatant") was withdrawn and frozen at -70'C. Unlabeled nuclei were isolated, incubated in similar fashion, recovered by centrifugation, resuspended in the "labeled supernatant," and incubated for 0, 5, 10, or 20 min. The reactions were stopped by the addition of 4 vol of ice-cold 0.88 M sucrose buffer, and the supernatant and nuclear acid-precipitable material were collected on Whatman GF/C filters, washed repeatedly, and counted as described. Alternately, Escherichia coli [3H]tRNA (Miles Laboratories, 43,gCi/ml, approximately 50,000 cpm/aliquot) was mixed in appropriate proportion with ATP, RNase inhibitor, and sucrose buffer; and unlabeled nuclei were resuspended in this incubation mixture. At 0 time or after incubation for 5, 10, or 20 min, the nuclei were sedimented by centrifugation, and the temperatures
nuclear and supernatant acid-precipitable material was measured as described. Statistical Analysis of Data. Linear regression coefficients were calculated by the method of least squares. Analyses were performed in each individual experiment and the results were grouped. RNA transport data at the various temperatures were homoscedastic. We tested the significance of fit as the F value of the ratio of explained variation to unexplained variation, or the corresponding t value. Analysis of covariance was performed on the calculated regression coefficients, and the significance of differences was tested with F values. RESULTS To investigate the energetics of RNA transport from isolated rat liver nuclei, we examined the temperature dependence of RNA in the temperature domain 35-00C. By following our standard protocol, we assessed transport after various incubation periods ranging from 5 to 20 min. When initial linear rates (up to 5 min) of ATP-stimulated transport were measured and used for Arrhenius analysis, a straight line was obtained and the activation energy for the RNA transport process was found to be 13 kcal/mol (1 cal = 4.184 J) (Fig. 1 and Table 1). However, when ATP-dependent transport obtained after a 20-min incubation was exhibited on an Arrhenius plot in the domain up to 370C, we observed two linear segments (one of positive slope and the other of negative slope) intersecting near 20'C. A significant decline in RNA transport (t20% of nuclear standard) was noted at 350C compared to that at 200C, resulting in a "peaked" graph reflecting maximal transport near 20'C. When 20-min transport data from the lower-temperature domain (20-0OC) were utilized in calculations (see Table 2), an activation energy similar to that derived from initial linear rates over the entire temperature domain was obtained, because RNA transport proceeded linearly in this domain (see
below). To ascertain the basis for the progressive decline
in
per-
centage of RNA transported in the upper-temperature domain (35-20° C) in the presence of ATP, we studied the time course
of RNA
transport. Transport was not linear over the 20-min incubation period in this domain; in general, the higher the temperature, the more rapidly maximal transport was observed. At 35'C, for example, 60.5, 57.5, 43.2, and 27% of the'acid-
precipitable nuclear RNA was present in the supernatant after 5-, 10-, 20-, and 30-min incubations (with ATP), respectively. These points were colinear (P
