Download - A set of non-histone proteins isolated from the nuclei of various rat tissues

Eur. J . Biochem. 135, 61 - 68 (1983) @) FEBS 1983

A set of non-histone proteins isolated from the nuclei of various rat tissues Akira INOUE, Youtaro HIGASHI, Tadayoshi HASUMA, Seiji MORISAWA, and Munehiko YUKIOKA

Department of Biochemistry, Osaka City University Medical School

(Received April 19, 1983) - EJB 83 0397

A set of non-histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat. Following moderate digestion of thoroughly washed nuclei with DNase 1 or micrococcal nuclease, EDTA was added to 5 m M to the reaction mixture and the preparation centrifuged. We found that the supernatant contained a limited amount of non-histone proteins (fraction SI). Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A - D) each possessing closely spaced doublets or a triplet. Their molecular weights were A, 76100-80000; B, 48200-49500; C, 44500-45200 and D, 39500-41 500. The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus. Two-dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges. Some were labelled with [32P]orthophosphate in vivo, or with [y-32P]ATP and purified nuclear protein kinase NII in vitro. The released proteins B -D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations a t similar sites in the nucleus. The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B - D constitute a group of proteins that have several common characteristics.

The abundance of protein species in the cell nucleus varies with the protein; some are structural or regulatory in function while others are catalytic in various biochemical processes. Isolation and characterization of these nuclear proteins should greatly aid in understanding the structure and function of the nucleus as well as the molecular properties of the protein in question. Isolation of a group of proteins which share molecular properties may be advantageous, as a common biological function may be involved. The precedent of this can be illustrated with histones and high mobility group (HMG) proteins. Histones, the building molecules of the nucleosomes [ I ] , can be readily extracted in a set from the chromatin with dilute mineral acids [2]; H M G proteins, which confer a structural characteristic of the active Chromatin [3] , are extracted as a group from chromatin with 0.35 M NaCl and remain soluble in 2

There have been numerous studies on various nuclear architectures such as chromatin, nuclear matrix and nuclear envelopes. One approach is to use DNA-hydrolyzing enzymes ; for example, DNase I has been used to distinguish the transcriptionally active from the bulk chromatin in differential digestion kinetics [5], or to isolate the nuclear pore complex- lamina fraction [6] and nuclear matrix [7].

In our studies on nuclear proteins we were interested in protein components liberated from the nuclei by digestion with DNA-hydrolyzing enzymes. We report here that a novel and remarkably simple set of non-histone proteins was isolated from the nuclei by nuclease digestion and that these proteins appear to be structural constituents of the chromatin.

trichloroacetic acid [4].

Ahhreviutions. SDS, sodium dodecyl sulfate; HMG proteins, high mobility group proteins.

En;yymes. DNase I (EC 3.1.21.1); ~nicrococcal nuclease (EC 3.1.31.1); protein kinase (EC 2.7.1.37); poly(ADP-ribose) polymerase (EC 2.4.99.-); DNA-dependent RNA polymerase (EC 2.7.7.6).

MATERIALS AND METHODS

Preparation of SI proteins

Rat liver nuclei were isolated through 9 tissue volumes (v/w) of 2.3 M sucrose, 3 mM MgC12, 0.2 mM phenylmethylsulfonyl fluoride, as described previously [8], and washed with 15 tissue volumes of 0.3 M sucrose, 3 m M MgCI2, 0.2 m M phenylmethylsulfonyl fluoride, 12 mM Tris/HCl, pH 7.6, by gentle stirring at 0 "C for 20 min followed by centrifugation at 3000 x g. The two supernatants (NS1 and NS2) were saved, and the pelleted nuclei, suspended in the same solution containing 0.5 m M CaClz at 30 AZbo units/ml, were incubated with DNase I (2.0-2.5 pg/ml, Sigma, DN- CL) or niicrococcal nuclease (25 units/ml, Worthington) at 30°C for a determined time. At the end of incubation the mixture was chilled on ice, EDTA added to make a final concentration of 5 mM (with 0.1 M solution), and the preparation centrifuged at 3000 x g for 10 min. The supernatant (SI) was saved, and the pelleted nuclei were suspended in 1 mM EDTA, pH 7.2, in the same volume as S1, left to stand for 20 min on ice with occasional agitation, and then centrifuged at 10000 x g for 20 min to separate into the second supernatant (S2) and the pellet (P) fractions. Fractions S1, NSI and NS2 were clarified by recentrifugation at 50000 x g. When fraction S1 alone was to be prepared, the reaction mixture after adding EDTA was centrifuged directly at 50000 x g. Extent of digestion was monitored by percentage solubility of the digested chromatin DNA in 7 % perchloric acid.

SDS/polyacrylamide gel electrophoresis

SDS/polyacrylamide gel electrophoresis was performed essentially as described by Laemmli [9] using 11 % or 13% acrylamide slab gels (dimensions: 2 x 150 x 150 mm). Pro- teins were precipitated with 2 vol. cold ethanol after making the solutions 0.1 M NaCI, and dissolved in the electrophoresis

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sample buffer (0.1 SDS, 1 % 2-mercaptoethanol, loo/, sucrose, 10 niM sodium phosphate, pH 7.2). Samples for electrophoresis were also prepared, in an alternative manner. 0.9 vol. 1 % SDS, 0.1 M sodium phosphate, pH 7.2, was added to the solutions, which were subsequently dialyzed against 0.002 % SDS, 0.2 mM sodium phosphate, pH 7.2, lyophilized, and dissolved in 10 % sucrose, 1 2-mercaptoethanol to make a final concentration of 0.1 % in SDS. M , was determined using bovine serum albumin (68 000), chicken egg albumin (43000), bovine pancreatic DNase I (31 000) and chymotrypsinogen (25 000) as standards. Bromophenol blue was added to each sample to serve as a tracking dye. After electrophoresis, gels were stained with 0.05 % Coomassie blue in 10 % acetic acid, 25 % isopropanol for 40 min, and destained by changing the destaining solutions; first 10 acetic acid with two changes, then 35 % methanol/7 % acetic acid, and finally 10 % acetic acid.

Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was performed according to Peters and Comings [lo] with the following modifications. Ampholytes (LKB) for isofocusing gels (3.2 mm diameter x 100 mm) were mixed at a volume ratio of 2 (pH 3-10): 2 (pH 6-8): 2 (pH 9-11): 1 (pH 5-7). Frac- tion SI (80 pg proteins for each gel) was dialyzed against 0.001 SDS, lyophilized, and dissolved in 0.01 vol. 0.050/0 SDS, 2.5 mM EDTA, 2.5 % 2-mercaptoethanol, 7 M urea, 3.75 % (w/v) ampholyte (pH 3 - lo), 6 % (v/v) NP-40 (sample buffer for isofocusing). Isofocusing was carried out in a cold box of 6 “C at a constant current of 0.35 mA/tube. When the voltage reached 400 V, electrophoresis was continued at this constant voltage for 17 h, and finally proteins were hyper- focused at 600 V for 1 h. pH measurements were made by slicing the extruded gels at 0.5 - 1 .O-cm intervals, followed by soaking of the gel pieces in 1 -ml portions of H20. SDS electrophoresis for the second dimension was carried out as described above.

Labelling of SI proteins by reductive methylation

SI proteins were labelled by reductive methylation, essentially according to the method of Rice and Means [Il l . S1 proteins (2.5 mg), collected by ethanol precipitation, were dissolved in 3 ml reaction buffer (6 M urea, 0.3 M NaCl, 5 mM Na borate, pH 9.0), precipitated with 3 vol. cold ethanol and centrifuged. This solubilization/precipitation step was repeated and the pellet was washed three times with ethanol and dried. To the proteins, redissolved in 3 ml of the reaction buffer and chilled on ice, 0.2 ml 1 mCi [3H]form- aldehyde (10 pmol) was added with stirring, and followed in 30 s by three 0.5-ml sequential additions of sodium boro- hydride (10 mg/ml H20). After 30 min, proteins were collected with ethanol, and washed successively as above by three solubilization/precipitation cycles and three times in ethanol. ”-labelled proteins were dissolved in 8 mM Tris/ HCl, pH 9.0, 0.1 mM phenylmethylsulfonyl fluoride and centrifuged at 2000 x g to remove insoluble materials. 85 % proteins were recovered at a specific radioactivity of 3.6 x lo3 counts min-’ pg-’.

Purification of protein kinuse NII

Rat liver protein kinase NII was purified from a 0.4 M NaCl nuclear extract by three successive column chroma-

tographies (DEAE-Sephadex, Bio-Gel A-3.5 m and phospho- cellulose) as previously described in [8], and finally by heparin- Sepharose chromatography’. The enzyme was more than 90 ”/, pure, as determined by SDS gel electrophoresis.

Preparative methods

Histones and HMG proteins were isolated from rat liver nuclei, as previously described in [I21 and [8], respectively. 40s ribonucleoprotein (RNP) particles, DNA and nuclear RNA were prepared from the isolated rat liver nuclei as described in [13], [I41 and [15], respectively.

Assay for enzyme activities

The enzyme activities of protein kinase, poly(ADP- ribose) polymerase and RNA polymerase I1 were assayed as previously described in [16], [17] and [I 81, respectively.

RESULTS

Digestion of nuclei with DNase 1

The nuclei were prepared from rat liver and washed ex- tensively in 0.3 M sucrose containing 3 mM MgClz to remove the soluble nucleoplasmic components. After digestion of the nuclei with DNase I, EDTA was added to 5 mM to the reaction mixture. Under these conditions the nuclear mem- branes remained intact, as determined by electron micro- scopy (not shown). The digest was centrifuged into the first (Sl) supernatant and the nuclear sediment, which was lysed in 1 mM EDTA, pH 7.0 and centrifuged again to yield the second (S2) supernatant and the pellet (P) fraction.

The appearance of DNA in S1 increased with time, amounting to 7.5 ”/, of total DNA in 10 min (Fig. 1 a). In S2, as much as 60% of total DNA was recovered. S1 DNA was completely soluble in 7 % perchloric acid, thus indicating a composition of fragmented smaller DNAs less than 20 base pairs long [5]. The recovery of proteins showed similar profiles to those of DNA. However, the protein in S1 (S1 proteins) was only 0.4 ”/, of the total nuclear proteins after 10 min digestion. This is in contrast to S2 proteins, which amounted to 55 ”/, at 10 min.

SDS gel electrophoresis revealed SI to be composed of a remarkably simple set of proteins resolvcd into four groups (A-D), and each possessing a singlet or closely spaced doublets (Fig.2a). We refer to them here as A, BI , B2 and so on, according to the mobility. Protein distribution between S1 and S2 was further examined using nuclear enzymes such as protein kinases, which are independent of cyclic nucleotides, or poly(AD-ribose) polymerase, which binds firmly to the chromatin. In contrast to high enzyme activities in S2, they were not detected in S1 (Fig.1b). Likewise, RNA polymerase I 1 activity was very high in S2, but nil in S1 (not shown). SDS gel electrophoresis of S2 and P demonstrated that these fractions comprised a variety of proteins (Fig. 2 b). Fraction S2 contained histones as major components together with various non-histone proteins of larger molecular weights. Fraction P contained a similar set of proteins, with non- histone proteins in a much higher proportion. If indeed S1 proteins are present, they are probably not major components in these fractions (Fig. 2b).

I Details will be described elsewhere (Y. Tei and A. Inoue, un- published).

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0 10 20 30 0 10 20 30 m

Time (rnin) Time ( m i d

Fig. 1. Digestion ofrat liver nuclei by DNase I : time course. (a) Release of nucleic acid and proteins into fractions S1 and S2. Suspensions of the thoroughly washed nuclei (30 A260/ml) were incubated with DNase I (2 pg/ml) at 30 "C. After the indicated periods of time, portions of the reaction mixture were withdrawn, EDTA added, and the preparation was centrifuged into the supernatant (fraction S1) and the pelleted nuclei. The latter were successively suspended in 1 mM EDTA and centrifuged to yield the second supernatant (S2) and the pellet (P) fractions. DNA (-) and protein (----) were determined for S1 and S2 and expressed as percentages of total nuclear contents. (0) Fraction S1; (0) fraction S2. ( x ) DNA rendered soluble in 7 % perchloric acid. (b) Distribution of nuclear enzyme activities in fractions S1 and S2. 5O-pl portions of each fraction were assayed for the activities of protein kinase (W, 0) or poly(ADP4bose) polymerase (v, 0). Incubation was performed in a total volume of 0.25 ml at 30°C for 10 min. Open and closed symbols denote enzyme activities in S1 and S2, respectively. DNA shown in (a) was also depicted as reference (0, 0)

The possibility that S1 proteins were those of the nuclear soluble fraction has to be considered. However, SDS gel electrophoresis clearly ruled out the presence of S1 proteins in the nuclear washes, NSl and NS2, indicating that S1 proteins are not those of the soluble nucleoplasm (not shown). Another control experiment was performed by omitting DNase I from the reaction mixture. Without the enzyme, S1 proteins released from the nuclei were almost totally protein A (Fig. 3, lane 2). Hence, proteins B-D are those liberated from the nuclei by the action of DNase I. The release of protein A without the enzyme was inhibited by inclusion of EDTA in the incubation mixture (Fig. 3, lane 3). Therefore, we assume that protein A may occur at the chromatin loci that have an extremely high sensitivity to some endogenous nuclease, the activity of which is suppressed by EDTA.

Fig. 2. (a) SDS/polyacrylamide gel electrophoresis of Sl proteins; (6) protein composition in fractions SI, S2 and P. (a) Rat liver nuclei were digested for 5 min with DNase I to give 10 % acid solubility. Fraction S1 was prepared, and the proteins (S1 proteins) analyzed by electrophoresis on a SDS gel (11 %, wjv, acrylamide). Faint bands, moving faster than group D, were sometimes observed when the samples were isolated after a prolonged handling time. (b) S1, S2 and P, prepared as described in the legend to Fig. l a , were analyzed for protein compositions by SDS gel electrophoresis (14 %, wjv, acrylamide). Lane 1, fraction S1 (12pg); lane 2, fraction S2 (48 kg); lane 3, fraction P (96 pg)

Characterization of SI proteins

a ) Molecular weights and nuclear content. The molecular weights of S1 proteins, determined by SDS gel electrophoresis, were 76100 (A), 49500 (BI), 48200 (B2), 45200 (CI), 44500 (C2), 41500 (DI) and 40200 (D2) (Fig.2a). B1 and B2, C1 and C2 and/or D1 and D2 occasionally produced fused bands, or proteins A and D were observed as a doublet and a triplet respectively, in different polyacrylamide gels. Faint bands moving faster than group D sometimes observed with different batches of samples. Since these faster moving bands became more discernible when samples were prepared after prolonged handling, proteolytic degradation may be involved. At present, we dot not regard these smaller proteins as S1 proteins. The yield of isolated S1 proteins was about 3.0 mg from 100 g of wet liver. By scanning the stained gel, the cor- responding yields of proteins A, B, C and D were estimated to be 0.22, 0.43, 0.56 and 1.79 mg, respectively. On the basis that 100 g liver yielded about 140 mg DNA, and that a single cell nucleus of the rat liver contains 8 pg DNA [19], we could roughly estimate the copy number of proteins A-D at 0.1 1 x lo6, 0.31 x lo6, 0 . 4 4 ~ lo6 and 1.53 x lo6 respectively,

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in a single cell nucleus. Since a selective extraction medium or an appropriate titration procedure which permits direct estimation of these proteins has yet to be designed, the content and copy numbers can probably be regarded as the lowest. Hence it is most likely that S1 proteins constitute structural components of the cell nucleus. For instance, high mobility group proteins, the nuclear structural proteins consisting of four species (HMG 1, 2, 14 and 17), were isolated at a yield of 3.5 mg from 100 g wet tissue. This indicates that S1 and HMG proteins are comparable in amounts which can be isolated under the present conditions. S1 proteins were compared by SDS gel electrophoresis with the well-characterized nuclear structural proteins, histones, HMG proteins and 40s RNP particle proteins (Fig. 4). 40s RNP particles were purified on a sucrose density gradient (Fig. 4 b, lane 4). The results clearly demonstrate that S1 proteins do not coincide with these proteins. Difference in mobility between histones and S1 proteins is also seen in Fig.2b. In addition, there are other nuclear proteins, with similar molecular weights. Tncluded are those of the nuclear envelope [20- 231, nuclear matrix [7] and scaffold [24], or the nuclear contractile proteins [10,25]. Their

Phoretic Patterns [7,10,20-251 revealed little Similarity to S1 Proteins. Therefore, we consider these s1 proteins to be newly found nuclear proteins, or at least their presence has

Fig. 3. Dependence of S1 protein liberation on DNase I action. Nuclear suspensions were incubated as described in the legend to Fig. 2a in the presence (lane 1) or absence (lanes 2 and 3) of DNase I. The sample in lane 3 was obtained by incubating the nuclear suspension in the presence of 5 mM EDTA. Portions (0.6 ml) of each S1 fraction were analyzed

weights '1 together with SDS gel

Fig.4. Comparison of Sl proteins with ( a ) histones and HMGproteins; ( h ) 40s RNPparticleproteins. (a) S1 proteins, histones and HMG proteins were isolated from rat liver nuclei, as described in Materials and Methods. The amounts of each protein applied on the SDS gel were equiva- lent to the wet tissue of 0.01 g for histones (lane l), 0.45g for S1 proteins (lane 2) and 0.44g for HMG proteins (lane 3), respectively. Acrylamide concentration of the gel was 13 %. (b) Ribonucleoprotein particles were extracted from rat liver nuclei and purified by sucrose density gradient centrifugation (35.0 ml 15 - 30% gradients in 0.1 M NaCI, 3 mM MgC12, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM triethanolamine/HCl, pH 8.0; 15 h run at 23000 rev./min, using a Hitachi RPS 27-1 rotor). The gradient was fractionated from the bottom into 23 fractions and proteins of each fraction were collected by precipitation with ethanol to be analyzed by SDS gel electrophoresis ( I 1 %, wjv, acrylamide). Lanes 1-5 are the fractions (fraction numbers 20, 16, 12, 8 and 2 respectively) of the gradient; lane 6, S1 proteins. The peak position of 40s RNP particles corresponds to the fraction 8 (lane 4)

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been demonstrated for the first time in nuclear digestion studies using a DNA-hydrolyzing enzyme.

6) Multiplicity of S l proteins. Further resolution of S1 proteins can be carried out by two-dimensional gel electrophoresis (Fig. 5 a). S1 proteins were run in an isofocusing gel for the first dimension and in a SDS slab gel for the second.

Table 1. Comparison of S l proteins with known nuclear structural proteins Molecular weights of various nuclear structural proteins are summarized. The S1 proteins depicted in Fig.4 did not coincide with histones, HMG proteins or with 40s RNP particle proteins. Except for scaffold proteins (HeLa cells), all the other proteins were from the rat liver. All values were determined by SDS/polyacrylamide gel electrophoresis

Proteins Molecular weights ( x Refer- ence

S1 proteins 41, 45,49, 76 (D, C, B, A) Envelope proteins

Pore complex-lamina 60, 67,70 (lamins) [20] 58, 67, 70 w1

Pore complex 66, 73, 78, 150 [221 68, 74, 78, 184 ~ 3 1

Matrix proteins 62, 66, 69 [71 Scaffold proteins 50-55, 65-70 ~ 4 1 Contractile protein” 43 - 45 (actin) [lo, 251

a Nuclear actin [lo, 251 has a molecular weight close to that of protein C (45 000) ; however, the extractability characteristics dif- fered (4 M NdCl [25] versus 5mM EDTA (SI proteins); S1 proteins precipitated at 0.2 M NaC1, Fig. 8b}, together with the different patterns on two-dimensional gels (a single spot around PI 6.1 with action [lo] versus multiple spots at PI 5.8- 8.2 with protein C, Fig. 5a).

When the reference S1 proteins were run in parallel on the right margin of the SDS gel, each group A-C was resolved here into doublets and group D into a triplet. The two- dimensional gel pattern is very characteristic in that most of these SDS bands were further resolved into as many as nine spots, according to their charges. Proteins A, and C2 are acidic and proteins B1, D1 and D3 basic, while others are spread in neutral pH regions. Although the subgroup A2 appeared as a single spot, electrophoresis on an isofocusing gel, favorable for separating acidic proteins, revealed four closely arrayed spots (not shown). The results for individual subgroups are summarized in Table 2.

The multiple spots, due to different charges, may arise from postsynthetic modifications. Among many possible

Table 2. Multiplicity of S l proteins

Groups Sub- PI region Distribution Number groups range of spots

PI units

A A- 1 5.0 1 A-2 5.2-5.65 0.45 4

B B-1 8.2-9.2 1 .o 3 B-2 6.6 - 7.6 1 .o 3

C c-1 7.2-8.2 1 .0 2 c -2 5.8 - 7.6 1.8 9

D D- 1 ‘8.2 - 8.7 0.5 2 D-2 6.6-8.3 1.7 9 D-3 7.6-8.6 1.0 3

5.0 6.0 6.5 7.0 80 9.0 I I I I I I

b

.O

0 0

0.

Fig. 5. (a) Two-dimensional gel electrophoresis of SI proteins. (h i Two-dimensional gel electrophoresis of S l proteins phosphorylated by protein kinclse N l l : Schematic representation of the autoradiogram. (a) 80 pg S1 proteins were electrophoresed on a first-dimensional isofocusing gel and then on a second-dimensional SDS gel, as described in Materials and Methods. Numbers on the top coordinate indicate the pH gradient formed in the first-dimensional gel. The second dimension was run in a SDS gel (1 1 %, wjv, acrylamide). Reference S1 proteins were run in parallel on the right margin of the SDS gel. (b) S1 proteins (0.39 mg) were incubated at 30°C for 15 min with the purified protein kinase NII (20 pl, 1 pg) in a total volume of 0.6 ml containing 15 pci carrier-free [y-”P]ATP, 12 mM MgCI,, 40 mM Tris/HCl, pH 7.8. After the reaction mixture was dialyzed against 0.001 y! SDS, it was lyophilized and dissolved in 50 pl sample buffer for isofocusing. The sample (80 pg) was subjected to two-dimensional gel electrophoresis as above. The proteins were visualized by staining with Coomassie blue, dried and autoradiographed. Closed spots represent those which coincided with the stained S1 proteins. Arrows indicate protein D2 subspecies labelled, in a separate experiment, with [32P]orthophosphate given to the rat intraperitoneally

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I D

Fig. 6. S1 protein liberation by micrococcal nuclease. The nuclei were digested at 30 "C for 5 min with micrococcal nuclease (25 units/ml) or DNase I (2.5 pg/ml). Under these conditions, 8.0% and 7.8% chromatin DNA was rendered soluble in 7 % perchloric acid by micrococcal nuclease and DNase I, respectively. S1 proteins, equiva- lent to 0.6 ml fractions S1, were analyzed by SDS gel electrophoresis (11 %, w/v, acrylamide). The densitometric scanning of the gel stained with Coomassie blue is shown. (-) S1 proteins released by micrococcal nuclease; (----) those released by DNase I

reactions, ADP-ribosylation should be considered. However, incubation of the isolated nuclei with [3H]NAD failed to label S1 proteins. When rats were given [32P]orthophosphate intraperitoneally and killed at 3.5 h, S1 proteins were labelled. Two-dimensional gel electrophoresis followed by autoradio- graphy revealed that three spots of the D2 subspecies under- went modification with 32P (Fig. 5 b). Furthermore, when S1 proteins were incubated with [p3'P]ATP together with highly purified protein kinase NII (a major protein kinase in the cell nuclei of most tissues [16]), the same spots as well as three additional spots of D2 proteins and three spots of C2 proteins were found to be phosphorylated by the enzyme (Fig. 5 b). These experiments indicate that phosphorylation reaction is responsible for the multiplicity of S1 protein subgroups. However, since other proteins were not phosphorylated, modification(s) besides phosphorylation, or micro- heterogeneity in peptide sequences also has to be considered.

c) Kinetics of SI protein liberation and aggregation properties. The nuclei were digested with micrococcal nuclease or DNase I to a similar extent of acid solubility (8.0% with micrococcal nuclease and 7.8% with DNase I). Gel electrophoresis demonstrated that micrococcal nuclease liberated the same set of S1 proteins as those of DNase I. The scanning patterns of the stained gel revealed that DNase I liberated about 20% more S1 proteins than micrococcal nuclease (Fig. 6) ; however, the differences were not significant. There- fore, S1 proteins are considered to be localized in the regions sensitive to both of the enzymes. Fig.7 shows the kinetics of S1 protein liberation where the nuclei were digested with DNase I for various periods of time. Proteins B, C and D were released and their relative molar ratios remained fairly constant during digestion (Fig. 7). This would suggest that proteins B - D originated from the sites with similar structures, or possibly from the same sites. S1 proteins tend to aggregate, and once collected from the reaction mixture by ethanol precipitation, dissolution in buffer solutions of neutral pH was difficult. The proteins could be redissolved in a basic solution (8 m M Tris/HCl, 0.1 mM phenylmethylsulfonyl fluoride, pH 9.0), however, when exposed to lower pH or salts, portions precipitated (Fig. 8), particularly in neutral

2.5 5.0 100 Time (min)

Fig. 7. Time course of Sl protein liberation: clo.~e resemblance between proteins B, C and D in nuclease liberation kinetics. The nuclear suspensions were digested with DNase I, as described in the legend to Fig. 2a, for increasing periods of time to give DNA acid solubilities of 1.6% (0 rnin), 5 % (2.5 rnin), 7.8% ( 5 min) and 12.8% (10 min). Portions (0.6 ml) of each S1 fraction were analyzed on a SDS slab gel ( I l x , w/v, acrylamide), and the stained gel was traced by densitometry scans. From the areas of each peak, the content of individual protein was estimated in terms of the relative proportion to total S1 protein at each digestion time. Note that the relative molar ratios between proteins B, C and D remained fairly constant. irrespec- tive of the digestion time

5.0 7.0 9.0 0 0.2 0.4 P H NaCl ( M )

Fig. 8. Effects o f p H and salt concentration on S l protein aggregation. S1 proteins (35 pg) labelled by reductive methylation with [3H]- HzCO were incubated for 30 min on ice in a final volume of 0.5 ml at various pH values or by changing concentrations of NaCI. After incubation, the mixtures were centrifuged at 27000 x g for I S min and the supernatants were successively mixed with 50 pl bovine serum albumin ( 5 mg/ml). Proteins were precipitated with 3 ml 20% of trichloroacetic acid, and collected on glass filters for radioactivity counting, as described in [12]. Values were expressed as a percentage of the total input of radioactivity. (a) Effect of pH on S1 protein aggregation. Buffer solutions used were 5 niM sodium phosphate (pH 4.5-7.0) or 8 mM Tris/HCl (pH 7.3-9.0) each containing 0.1 mM phenylmethylsulfonyl fluoride. (b) Effect of NaCl concentration on S1 protein aggregation. S 1 proteins were exposed to various concentrations of NaCl in 8 mM Tris/HCl, 0.1 mM phenylmethylsulfonyl fluoride, pH 9.0

and acidic solutions (Fig.8a) and in solutions of higher salt concentrations (Fig. 8 b). The latter result suggests that the isolated S1 proteins have a hydrophobic property. Protein A preferentially precipitated ; whereas in the case of proteins B - D, 50 - 75 precipitated. Pelleted B - D showed com- positional ratios resembling those remaining in the supernatants (Fig. 9). Therefore, these proteins either have very

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Fig. 9. Sl protein compositions in the precipitated and the supernatant fractions. (a) Precipitation with salt. S1 protein solution (50 pg in 100 p1 8 mM Tris/HCl, 0.1 mM phenylmethylsulfonyl fluoride, pH 9.0) was made 0.2 M NaCl with 2 M solution, left on ice for 30 min and then centrifuged at 16000 rev./min for 20 min. Aliquots (one-fourth) of the supernatant and the pellet samples were analyzed by SDS gel electrophoresis. Lane 1, proteins remaining in the supernatant; lane 2, proteins spun down to the bottom of the centrifuging tube. (b) Precipitation at a lower pH. pH of the Sl sample (50 pg in 100 pl 8 mM TrisiHC1, 0.1 m M phenylmethylsulfonyl fluoride, pH 9.0), was lowered to 6.3 by addition of 11 p10.25 M sodium phosphate, pH 4.5. After 30 min on ice, the mixture was processed in a manner similar to (a). Lane 1, proteins in the supernatant; lane 2, precipitated proteins

similar solubilities, or more likely they interact to the point of aggregation.

d ) Occurrence of’s1 proteins in various tissues. When S1 fractions were prepared from rat liver, testis, spleen and brain, we found that all these tissues contained remarkably similar sets of S1 proteins (Fig. 10). Although close examination of the stained patterns revealed slight differences between different tissues with regard to electrophoretic mobility and com- positional ratio of the proteins, we postulate that sets of proteins similar to liver S1 proteins are present probably in various tissues as constituents of the cell nucleus.

DISCUSSION

When rat liver nuclei were digested with DNase I or with micrococcal nuclease, a set of proteins (S1 proteins A - D) was identified. S1 proteins were characteristic in that (a) they consisted of a remarkably simple set of proteins, as seen in SDS gel electrophoresis (Fig. 2a), (b) when they were subjected to two-dimensional gel electrophoresis, each of the SDS gel bands was further resolved into multiple spots dif- fering in charge (Fig. 5 , Table 2), and (c) S1 proteins seemed to interact and aggregate (Fig. 8 and 9).

The estimated nuclear contents indicated that these proteins are structural components of the cell nucleus. However,

Fig. 10. Presence of S l proteins in various tissues of the rat. Nuclei were isolated from various tissues as described in Materials and Methods, except for the brain where four tissue volumes of 2.3 M sucrose, 3 mM MgCI2, 0.2 mM phenylmethylsulfonyl fluoride were used. The nuclei were digested with DNase I at 30°C for 3 min, at which time acid solubility attained 5.6, 3.0, 4.6 and 4.7 with liver, spleen, brain and testis tissues, respectively. Lanes 1 - 4 represent S1 proteins obtained from the tissues in the above order; lane 5, S1 proteins from liver. Samples on lanes 1-3 and 4-5 were run on different gels

they did not coincide with the nuclear structural proteins that have already been isolated and characterized (Fig. 4, Table 1). It should be pointed out that these proteins may have en- zymatic and/or regulatory functions, although it seems un- likely since their nuclear contents are significantly high.

The digestion conditions we used were similar to those of Bloom and Anderson [26,27], who demonstrated that micrococcal nuclease preferentially digested the transcriptionally active chromatin and that the fragmented chromatin was released from the nuclei into the first supernatant [26]. Ac- cordingly we looked for a more complex set of chromatin proteins in the S1 fraction. We found that EDTA, which was added at the end of digestion, precipitated histones and many minor non-histone proteins of the S1 supernatant and left only proteins A - D in the solution’ (detailed results on this selective S 1 protein extraction will be described elsewhere). Proteins A ~ D and nucleic acid constituted 20 ;( and 50 of total protein or nucleic acid, respectively, in the supernatant obtained without addition of EDTA. Thus, proteins A - D are considered to possess a common property : they behaved in a similar manner toward the surrounding solutes.

The kinetics of liberation of proteins B, C and D suggested that they originated from the sites with similar structures in the nucleus (Fig. 7). Although protein A did not always share properties with other S1 proteins, these kinetic data together with the aggregation property and the solubility in 5 mM EDTA indicate that S1 proteins or at least proteins B, C and D constitute a group of proteins that have several common properties.

When digestion with DNase I was performed in RSB buffer (20 mM Tris/HCl, pH 7.4,lO mM NaCl, 3 m M MgCl’), which is used to cleave preferentially the active genes at 10 % acid solubility [5] , an identical set of S1 proteins was isolated (not shown). In addition, the conditions of digestion closely

* In the study of Bloom and Anderson 1261, addition of EDTA - ~~

has been omitted upon isolation of the first supernatant fraction.

68

resembled those of Bloom and Anderson [26,27]. Therefore, S1 proteins may be localized at specific sites, such as those of the chromatin or the nuclear matrix that contains active genes. In this context it is interesting that protein kinase NII phosphorylated some of the S1 proteins (Fig.5b), since this enzyme may possibly be involved in gene regulation: the enzyme phosphorylates and activates RNA polymerase I [28], and phosphorylates HMG proteins [S], which confer the structural characteristics on the active chromatin [3,29].

As shown in Fig. 10, S1 proteins were identified in various tissues of the rat, and this common occurrence strongly suggests the presence o f S1 proteins in other tissues as well. We identified a set of non-histone proteins in the cell nuclei, and these components could be readily isolated by digestion of the nuclei with DNA-hydrolyzing enzymes.

We thank Prof. Y. Tashiro and Dr S. Matsuura (Kansai Medical University) for electron micrographs of the cell nuclei, M. Uda for preparation of the gels and secretarial services and M. Ohard for critical reading of the manuscript.

REFERENCES 1. Bradbury, E. M., Maclean, N. & Matthews, H. R. (1981) DNA,

Chromatin and Chromosomes, Blackwell Scientific Publica- tions, London and Boston.

2. Johns, E. W. (1971) in Histones and Nucleoproreins (Phillips, D. M. P., ed.) pp. 2-45, Plenum, New York.

3. Weisbrod, S., Groudine, M. & WeintrdUb, H. (1980) Cell, 19,

4. Goodwin, G. H., Sanders, C. &Johns, E. W. (1973) Eur. J . Bio-

5. Weintraub, H. & Groudine, M. (1976) Science (Wush. D C ) 193,

6. Aaronson, R . P. & Blobel, G. (1975) Proc. Natl Acad. Sci.

7. Berezney, R. & Coffey, D. S. (1977) J . Cell Biol. 73, 616-637.

289 - 301.

chem. 38, 14- 19.

848 - 856.

USA, 72, 1007-1011.

8. Inoue, A,, Tei, Y., Hasuma, T., Yukioka M. & Morisawa, S.

9. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. (1980) FEBS Lett. 117, 68-72.

10. Peters, K. E. &Comings, D. E. (1980) J. CellBiol. 86, 135-155. 11. Rice, R. H. & Means, G. E. (1971) J . Biol. Chem. 246,831 -832. 12. Hasuma, T., Yukioka, M., Nakajima, S., Morisawa, S. & Inoue,

13. Karn, .I., Vidali, G., Boffa, L. C. & Allfrey. V. G. (1977) J . B id .

14. Suzuki, Y., Gage, L. P. & Brown, D. D. (1972) J . Mol. Biol. 70,

15. Mishima, Y., Kominami, R. & Muramatsu, M. (1981) Taisha

16. Yutani, Y., Tei, Y., Yukioka, M. & Inoue, A. (1982) Arch.

17. Yukioka, M., Okai, Y., Hasuma, T. & Inoue, A. (1978) FEBS

18. Yukioka, M., Hatayama, T. &Inoue, A. (3982) J . Mol. Biol. 155,

19. Leuchtenberger, C., Vendrely, R. & Vendrely, C. (1951) Proc.

20. Gerace, L., Blum, A. & Blobel, G. (1978) .I. Cell Biol. 79, 546-

21. Richardson, J. C. W. & Maddy, A. H. (1 980) J . Cell Sci. 43,

22. Krohne, G., Franke, W. W. & Scheer, U. (1978) Exp. Cell Res.

23. Lam, K. S. & Kasper, C. B. (1979) J . B id . Chem. 254, 11713-

24. Adolph, K. W. (1980) J . Cell Sci. 42, 291 -304. 25. Douvas, A. S., Harrington, C. A. & Bonner, J. (1975) Proc.

Nut1 Acad. Sci. USA, 72, 3902- 3906. 26. Bloom, K . S. & Anderson, J. N. (1978) Cell, 15, 141-150. 27. Bloom, K. S. & Anderson, J. N. (1979) 1. Bid . Chem. 254,

28. Ducenian, B. W., Rose, K. M. & Jacob, S. T. (1981) J . B id .

29. Weisbrod, S . & Weintraub, PI. (1979) Pro(.. Nut1 Acad. Sci. USA,

A. (1980) Eur. J . Biochem. 109, 349-357.

Chem. 252,7307 - 7322.

637-649.

(Metabolism} 17, 79 - 88 (Japanese).

Biochem. Biophys. 218, 409-420.

Lett. 86, 85 - 88.

429 - 446.

Nut1 Acad. Sci. USA, 37, 33 - 38.

566.

253 - 267.

116,85- 102.

11 720.

10532- 10539.

Chem. 256, 10755- 10758.

76, 630- 634.

A. Inoue, Y. Higashi, T. Hasuma, S. Morisawa, and M. Yukioka, Department of Biochemistry, Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka-shi, Osaka-fu, Japan 545