A major soluble acidic protein located in nuclei of diverse vertebrate species

Copyright @ 1980 by Academic Pres, Inc. All rights of reproduction m any form reserved 0014-4827/80/090167-23$02.00/O

Experimental

A MAJOR SOLUBLE

Cell Research

ACIDIC

OF DIVERSE

PROTEIN

VERTEBRATE

GEORG KROHNE Division of Membrane

Research

LOCATED

IN NUCLEI

SPECIES

and WERNER W. FRANKE

Biology and Biochemistry,

German Caccer

129 (1980) 167-189

Institute of Experimental

Center, D-6900 Heidelberg,

Pathology,

Germany

SUMMARY Guinea pig antibodies against the most frequent nuclear protein of oocytes ofxenopus laevis were used for the characterization and localization, by immunoprecipitation and immunofluorescence microscopy, of this or immunologically related protein(s) in tissues and in cultured cells of diverse vertebrates. The antigen present in Xenopus oocyte nuclei was soluble, acidic, and contained polypeptide(s) of molecular weight of about 30000. It appeared with a broad range of isoelectric variants (apparent isoelectric point (IEP) values between 4.7 and 6.0) which was shown to reflect at least partly differences of phosphorylation. A similar protein was also abundant in oocytes of other amphibia but distinct differences in electrophoretic mobility and IEPs were noted between different species. Similar or immunologically related proteins were also detected, by immunofluorescence microscopy, in diverse tissues and cultured cells of other vertebrates (amphibia, birds, mammals). Mammalian cell lines that showed strong nuclear staining with antibodies to the protein of Xenopus oocytes included examples of marsupial (PtK2), murine (mouse 3T3), and human (HeLa) origin. Certain cell types characterized by very low levels of transcription and predominance of condensed chromatin, such as amphibian and avian erythrocytes and late stages of spenniogenesis, appeared to be devoid of the protein. During interphase and meiotic prophase the protein was detected only in the nucleus where it showed a dispersed distribution, sometimes in ‘punctate’ patterns, and no specific enrichment in nucleoli or dense chromatin structures. During nuclear divisions of both mitosis and meiosis the protein was spread throughout the cytoplasm and appeared to be greatly reduced, if not totally absent, in metaphase and anaphase chromosomes. In immunolocalization experiments considerable losses of the protein into the incubation solutions were noted, which could give rise to artifacts of localization, but this effect could be reduced by modifications of preparation conditions. Possible functions of this protein, the determinant of which has been largely conserved in vertebrate evolution and which seems to be similar, if not identical to the ‘nucleosome assembly factor’ protein described in egg homogenates of Xenopus, are discussed, especially in relation to the formation of chromatin and ribonucleoproteins.

For the determination of the intracellular location of a protein two methodical approaches have been primarily taken, i.e. fractionation of cellular components and localization in situ by the use of specific reagents, including antibodies against the protein. Both methods have their specific disadvantages and problems of interpretation. In cell fractionation, for example, the questions of cross-contamination and selective loss of components during the prepara-

tion are critical, whereas in localization techniques the specificity of the reagent and the preservation and accessibility of the protein is crucial. As to the proteins of the cell nucleus, components stably bound to sedimentable nuclear structures have been most intensely studied with both methods, obviously since these proteins usually are not removed or translocated to any great extent during either cell fractionation or preparation for localization in situ. ExExp Cdl Res 129 (1980)

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amples include the specific nuclear localization of (i) chromatin-bound proteins such as histones and RNA polymerases (for review see [l-4]); (ii) ribonucleoprotein components [5, 61; (iii) proteins associated with gel meshworks and filaments (for review see [7]); and (iv) insoluble karyoskeletal components (/g-11]; see there for further references). On the other hand, knowledge of less stably associated nuclear proteins (‘soluble proteins’, diffusible proteins, readily extracted proteins) is still very poor, primarily due to the technical limitations mentioned above. Studies involving nuclear isolation in non-aqueous media have shown that a large number of soluble enzymes, including those of the glycolytic pathway, are located in nuclei, and that some proteins seem to be exclusive to the nucleus [ 12, 131; see’ there for further references). Nuclear transfer and cytoplasmic injection experiments have shown that certain soluble proteins are distributed between nucleus and cytoplasm whereas others are accumulated entirely within the nucleus ([14-231; for nucleocytoplasmically ‘shuttling’ proteins see [23, 241). We have recently described that the most abundant nuclear protein of the oocyte of the frog, Xenopus laevis, is a soluble, acidic, strongly phosphorylated protein of approximate molecular weight (M,) of 30000 which by immunoprecipitation and by immunofluorescence microscopy has been found highly concentrated in, if not exclusive to, the nucleus of the oocyte as well as of other cells of Xenopus [25]. This protein is readily lost from isolated nuclei and nuclear structures during incubation in aqueous solutions as well as during incubation of cells and tissues with antisera and buffers used for immunofluorescence microscopy. In the present study we demonstrate, .using immunological techniques, that a similar, imExp CeNRes 129(1980)

munologically cross-reacting protein is also present in nuclei of oocytes of other amphibian and avian species, as well as in a broad range of cells of tissues and cultured cells of amphibia, birds and various mammals. This protein, however, has not been detected in nuclei of certain transcriptionally inactive cells such as amphibian and avian erythrocytes and late spermatids and spermatozoa. During the immunological localization of this protein in cultured cells and sections of frozen tissues we have noted that dramatic losses of this protein can occur under commonly used incubation conditions, resulting in artificial localization, and we have learned how important the choice of the procedure is in the correct immunolocalization of soluble proteins.

MATERIAL C&and

AND METHODS

tissues

Small pieces of ovaries were removed from anaesthetized amphibians [cf 93 of the following species: Xenopus laevis, Rana pipiens, R. esculenta, Bufo bufo. Bombina variepata. Pleurodeles waltlii. Triturus crhthtus, and T. aliestrh. Oocyte proteins’ were la-

belled with [aH]leucine (400 @i/ml, 110 Ci/mmol) or [3sS]methionine (300 $X/ml, 800 Ci/mmol; all from Radiochemical Centre, Amersham) by incubation of ovary pieces at 21°C for 16-20 h in modified Barth’s solution [26]. Small tissue pieces (ovaries, testes, liver, skeletal muscle) from the diverse amphibians, from chicken and from rat were frozen and processed for freezesectioning as described [lo]. Xenopus laevis epithelial kidney cells, embryonic chicken fibroblasts, rat kangaroo cells (PtKJ, mouse 3T3 cells, and HeLa cells were grown on coverslips as described [27].

Isolation of oocyte nuclei and treatment of nuclearproteins with alkaline phosphatase Nuclei and nuclear contents of vitellogenic oocytes were manually isolated in 83 mM KCI, 17 mM NaCI, 10 mM Tris-HCI (pH 7.2), collected in 96% ethanol, and stored at -20°C [9,25]. For each experiment with alkaline phosphatase (grade I, from calf intestine; Boehringer, Mannheim) 3UO nuclei fixed in ethanol were dried under nitrogen, and proteins were then

Localization solubilized by incubation of the dried sediment for 30 min at 20°C with 30 LLI0.1 M Tris-HCI buffer (pH 8.5). Non-soluble components were sedimented (4 min at 8 CKlOa). The sunematant was adiusted with 0.1 M Mg&to 5 mM &Cl,, 5 units (2 4) of alkaline phosphatase were added, and the mixture was incubated for 30 min at 37°C. The reaction was stopped and the proteins precipitated by addition of 4 vol of ice-cold acetone.

Gel electrophoresis Onedimensional slab gel electrophoresis was performed essentially as described by Laemmli ([28]: 10 % or 12% gels) or Thomas & Komberg ([29]: 15% or 16% aels). Two-dimensional gel electrophoresis was done as described by O’Farreli[30] with the modifications of De Robertis et al. [31]. Ampholine buffers (Servalyt, Serva, Heidelberg)-allowing optimal separation of uroteins in a pH range from 4 to 7 were used. Ethanol-fixed and nitrogen-dried nuclei were solubilized in lvsis buffer without digestion of nucleic acids by DNA& and RNAse. The&soluble components were sedimented (4 min at 8000 g) and analysed on one-dimensional gels. Only 50% of total nuclear protein but all of the nrotein with aouarent molecular weight (MT) of 30060 was soluble ;n the buffer used for two-dimensional electrophoresis [cf 321. Gels were stained with Coomassie blue or processed for autoradiofluorography [33]. For mol. wt determination the following reference proteins were used: myosin heavy chain (200000), phosphorylase-a (94000), bovine serum albumin (67000), rabbit muscle actin (42000), and chymotrypsinogen (25 000).

Antisera

and antibody preparation

Antibodies were raised in guinea pig following the procedure and booster regime described [34]. 1200 ethanol-precipitated nuclear contents (for polypeptide pattern, see fig. 1, slot 4) were taken for the first immunization and 600 nuclear contents for the booster injection at day 21. In another experiment the polypeptide band of M, 30000 was cut out of the gel, the protein was eluted, precipitated with acetone and used as antigen (for description of the method see ref. [27]). The immunoglobulin (IgG) fraction was prepared by the chromatography of the serum on DEAE-cellulose [cf 351. Monospecific antibodies were obtained by dissociation of direct immunoprecipitates (for preparation of direct immunoprecipitates, see next chapter) for 15 min in 4 M MgC&, 10 mM Tris-HCl (pH 6.8). Antibodies against histone H2B and preimmune sera were used as controls (for details, see [lo]).

Immunodiffusion and immunoprecipitation Immunoreplica tests were done as described [36; cf 27, 371.For immunoprecipitation the proteins of 60 oocyte

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nuclei were solubilized, either directly after nuclear isolation or after storage in %% ethanol, in 30 ~1 of phosphate-buffered saline (PBS: 137 mM NaCl, 7 mM Na,HPG,, 1.5 mM KH*PO,, 2.7 mM KCI). Supernatants obtained after centrifugation (4 min at 8 000 g, or 30 min at 120000 g) were mixed with 10 ~1 antiserum against the M, 30000 protein and incubated for 45-60 min at 21°C. The direct immunoprecipitates were collected by sedimentation (4 min at 8 000 g) and washed 2-3 times with 100~1 PBS. For collecting antibodv-antieen comolexes not pelleted at low speed the 8 & g sugematanis obtained-were mixed with 5-8 mg nrotein-A-Seoharose CL-4B (Pharmacia, Uppsala). ‘After additional incubation for 45 min at 21°C the IgGprotein A complexes were pelleted (5 min centrifugation at 500 g) and washed twice with PBS.

Indirect immunofluorescence microscopy Frozen sections and cells grown on coverslips were processed for indirect immunofluorescence microscopy as described [lo, 271. For optimal staining of sections and cells grown on coverslips different fixation procedures and incubation times were examined. (1) Air-dried sections (dried for 2-48 h) were fixed for 15 min at -20°C with acetone and were then again airdried. (i) Sections thus dried were incubated for 45 min at 20°C with antiserum (1: 20 or 1 : 30, diluted with PBS) or IgG (20-50 pg/ml, in PBS), washed three times for 10 min each with PBS, then incubated for 45 min with the FITC-conjugated rabbit-anti-guinea pig (or goatanti-rabbit) antibodies (0.2-0.5 mg/ml, in PBS; MilesYeda, Rehovot, Israel), washed again three times for 10 min each, dehydrated in 95% ethanol, and embedded in Elvanol or Moviol4-88. (ii) Dried sections were incubated for 10 min at 20°C with antiserum or IgG solution, washed twice for 5 min each with PBS, then incubated with PBS containing the ‘second antibody’ for 10 min, followed by two washes in PBS for 5 min each, dehydration and embedding. (iii) Sections were incubated for 10 min at 20°C with antiserum or IgG preparation in 100mM MgCI,, 10mM Tris-HCl (pH 7.4). Then the sections were washed twice for 5 min each with PBS and incubated with the ‘second antibody’, in the absence of MgCl,, as described above (1 ii). (2) Air-dried sections (2-48 h) were fixed for 10 min with 2 % formaldehyde in PBS freshly made from paraformaldehyde. The sections were then washed for 1 min in PBS, fixed with acetone at -20°C for 15 min and airdried. Sections were incubated with antisera or IgG as described above (1 ii). (3) Cultured cells grown on coverslips were processed as follows. (i) Cells were fixed for 5 min at -20°C with methanol, dipped three times in acetone at -2O”C, and then airdried. The coverslips were incubated with antisera or IgG for 45 min as described under 1i and washed only once or twice for 5 min each after incubation with antibodies. Exp Cd Res 129 (/980)

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Fig. I. Gel electrophoresis (12 % gel, slots l-3) of total nuclear proteins (from 25 Xenopus oocytes) labelled with [W]leucine seen after staining with Coomassie blue (slot 2) and autoradiofluorography (slot 3). Reference proteins (slot 1) are, from top to the bottom, myosin heavy chain, phosphorylase-a, bovine serum albumin, actin, chymotrypsinogen. Arrows denote the polypeptide band of Af, 30000. Characterization of guinea pig antibodies to this protein is shown by immunoreplica (see slots 4 and 5) and immunoprecipitation (see slots 6-i-10). In the sample shown in slots 4 and 5 the proteins of 60 nuclear contents (slot 4, Coomassie blue) of Xenopus oocytes were separated on a 10% polyacrylamide gel; in the corresponding agarose overlay immunoreplica (slot 5, on a parallel slot containing the proteins from 90 oocyte nuclei; Coomassie blue-stained) only this polypeptide of M, 30000 has formed a precipitate with the antiserum used. Direct immunoprecipitates (12% gel, slots 6-10, Coomassie

blue-stained) using protein of 60 oocyte nuclei from Xenopus (slot 7) and Rana pipiens (slot 9) were obtained after mixing the solubilized (non-sedimentable) nuclear proteins with the antibodies against the M, 30000 protein. Direct immunoprecipitates obtained (slot 7; note the minor contamination by some serum albumin) ate shown as well as immunoprecipitates that have been washed after sedimentation three times with PBS (slot 9). Non-sedimentable antigen-antibody complexes from Rnna pipiens oocyte nuclei (supernatant of the first immunoprecipitate pellet shown in slot 9) have been collected by binding to protein-A-Sepharose (pellet therefrom is shown in slot 10). Slots 6 and 8 show the total nuclear proteins of oocytes from the same frogs (Xenopus: slot 6, 25 nuclei; Rana pipiens: slot 8, 20 nuclei) for comparison. The arrows denote the position of the protein with M, 30000; asterisks denote heavy, brackets light chains of guinea pig immunoglobulins (IgG).

(ii) Cells were incubated for 10 min with antisera or IgG and washed for 5 min after each mcubation. (iii) Cells were first fixed with 2 % formaldehyde (in PBS) for 10 min and then processed as described under 3 ii.

Specimens were observed and photographed using a Zeiss Photomicroscope (Zeiss, Oberkochen, FRG) equipped with epifluorescence illumination using various oil immersion objectives (16x, 25x, 40X, 63x).

Exp CeNRes 129 (1980)

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trophoresis a prominent polypeptide band of apparent molecular weight (M,.) of 30 000 (‘M, 30000 protein’) was observed (figs l-3) which represented 9-10% of the total protein stained [cf 253. This protein component was the most abundant protein present in these nuclei and exceeded, in freshly isolated nuclei, the protein contained in the band with an apparent M, 42000 which included nuclear actin (figs 1-3; for oocyte nuclear actin cf [7, 22, 381; as to the presence of some other minor polypeptides in this band see fig. 4a and ref. [32]). Incubating living oocytes in [3H]leucine and [35S]methionine showed that this protein was continuously synthesized (fig. 1, slot 3) and incubating oocytes in [32P]phosphate demonstrated that this protein is highly phosphorylated (for data see [25]). When isolated oocyte nuclei or gelified nuclear contents were incubated for prolonged periods of time in various buffers, including some of physiological ionic strength, a gradual decrease of this protein was observed (e.g., fig. 1, slot 4), which reflected its readiness to extraction and solubilization (for details see [25]; see also the data of ref.

[391). Fig. 2. Gel electrophoretic comparison (12% gel) of total nuclear proteins of oocytes from three different frog species. Slot 2: Xenopus kzevis, 25 nuclei; slot 3: Rana esculenta, 23 nuclei; slot 4: Rana pipiens, 2.5 nuclei. The arrow denotes the position of the protein of apparent M, 30000; bars denote some of the polypeptides significantly different between the three anurans. Note that the M, 30000 protein is prominent in ah three species. Reference proteins (slot 1) are as in fig. 1.

RESULTS Characterization of the antigen protein present in amphibian oocyte nuclei by gel electrophoresis and immunoprecipitation

When total proteins of oocyte nuclei of Xenopus laevis were separated on gel elec-

Guinea pig antibodies to this protein only precipitated material of this polypeptide band as shown by immunoreplica gels (e.g., fig. 1, slot 5), which also indicated that this protein was not a breakdown product derived from other larger proteins. The high specificity of the antibodies was also noted in immunoprecipitates from solution in buffers of physiological ionic strength (fig. 1, slots 6 and 7). Examination of the immunoprecipitates obtained by two-dimensional gel electrophoresis further demonstrated that the antibodies reacted with the whole range of the various isoelectric variants of this protein and were not selective for special subclasses (for data see [25]). The antiExp Cell Res 129 (1980)

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Fig. 3. Gel electrophoretic

separation (16% gel) of total nuclear proteins of oocyte nuclei from different amphibians (slot 2: Xenopus, 23 nuclei; slot 3: Bufo bufo, 15 nuclei; slot 4: Pleurodeles waltlii, 22 nuclei? and the direct immunoprecipitates (60 oocyte nuclet each) obtained after mixing the solubilized nuclear proteins with the antiserum against the M, 30000 protein (slot 5: Xenopus; slot 7: Bufo bufo; slot 9: Pleurodeles waltlii). The non-sedimentable antigen-antibody complexes*of each supematant were collected by binding

to protein-A-Sepharose beads and are shown in slots 6 (Xenopus), 8 (Bufo) and 10 (Pleurodeles). Reference proteins (slots 1, 11) are, from top to the bottom, phosphorylase-a, bovine serum albumin, actin, chymotrypsinogen. The M, 30000 protein is labelled by short white bars (slots 24) and arrows (slots 5-10); positions of IgG heavy (H) and light (L) chains are denoted by arrowhead and bracket, respectively. Note separation of different electrophoretic mobility subgroups in light chains of IgG.

bodies to the M, 30000 protein from isolated germinal vesicles of Xenopus laevis also specifically precipitated this protein from the soluble proteins remaining in supematants obtained after heat-denaturation of total proteins of(i) isolated oocyte nuclei (see [25] for conditions of heat treatment and centrifugation); of (ii) homogenates of unfertilized eggs; and of (iii) whole ovaries of Xenopus (not shown here; for data see

These antibodies also specifically precipitated one polypeptide band of about 30000 M, from total proteins of oocyte nuclei of other anurans (fig. 1, slots 8-10, and fig. 3, slots 7 and 8) and an urodelan (e.g., fig. 3, slots 9 and lo), thus indicating that this protein is not species-specific. In all amphibian species examined the M, 30000 protein was the most prominent polypeptide band (fig. 2), always representing more than 8% of total nuclear protein as esti-

r3m Exp Cell Res 129 (1980)

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IEF

IEF

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’

t

I

b

I

I

Fig. 4. Two-dimensional gel electrophoresis (15 % gel in second dimension) of oocyte nuclear proteins from an anuran, Xenopus (a, from 33 nuclei) and from a urodelan species, Pleurodeles (b, from 32 nuclei). The total range of isoelectric variants of the M, 30000 polypeptide(s) is indicated by the brackets; the major spot components are denoted by the arrows. The arrowhead denotes the position ‘of nuclear actin. The vertical scale presents pH values for both gels in the

isoelectric focusing direction (IEF, from top tobottom: pH 7.0, 6.0,5.0,4.5), the horizontal bars at the bottom indicate apparent M, values in the SDS-containing gel (from left to right: 1OOOtMand 30000). Note that the M, 30000 protein is a major nuclear component in oocytes of both species but that the polypeptide(s) present in Pleurodeles in general have lower electric charges and electrophoretic mobility.

mated from densitometry of proteins separated on gels and stained with different dyes (for details see [25]). Comparison of total nuclear proteins of oocytes from different species showed that this protein is indeed a consistently predominant nuclear protein, present in similar amounts, in contrast to numerous other nuclear proteins and polypeptides, respectively, which exhibited conspicuous differences from species to species (figs 2 and 3, slots 2-4). For example, we found great quantitative differences in the actin-containing polypeptide band, even among related anuran

species (compare fig. 2, slots 24, and fig. 3, slots 2-4). Interestingly, however, slight but significant differences in the gel electrophoretic mobility of the M, 30000 protein were noted between various amphibian species (e.g., Xenopus laevis, Bufo bufo, and Pleurodeles waltlii: fig. 3, slots 2-4 as well as slots 5, 7, and 10). In urodelan species the differences to the M, 30000 protein of Xenopus oocytes were more pronounced: for example, in Triturus alpestris and Pleurodeles waltlii, this protein was clearly of lower electrophoretic mobility (fig. 3, slot 4), equivalent to an apparent E.rp Cd

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IEF , #

,b 1 Fig. 5. One-dimensional (a; 16% gel) and two-dimen-

sional (b, c; 15% gels) gel electrophoresis of nuclear proteins from Xenupus oocytes obtained with (a, slot 2; c) and without (a, slot 1; b) treatment with alkaline phosphatase (a, slot 3: alkaline phosphatase alone). For each experiment the solubilized proteins of 33 nuclei (a: slots 1, 2) or 40 nuclei (b, c) were used. After treatment with alkaline phosphatase the M, 30000 protein usually reveals splitting into two bands (a. slot 2, denoted by the bars). The bracket in b indicates the total range of the isoelectric variants of the M, 30000 protein, the larger arrows in b and c denote the main isoelectric ‘spot’ components seen before (b) and after

I

c

incubation with the phosphatase (c, smaller arrows denote minor spot components which are not always observed). The arrowheads denote the position of nuclear actin, the bracket in c shows the position of alkaline phosphatase (AP). The vertical scale presents the pH values for both gels (from top to bottom: 7.0, 6.0,5.0, 4.9, the horizontal bars indicate the apparent molecular weights (from left to right: 100000 and 30000). We cannot exclude, however, that the dephosphorylation has been incomplete and that some deamidation and/or carbamylation during the procedure has contributed to the appearance of these polypeptides.

M,. of 32000 (fig. 3, slots 4 and 10, and fig. 10). Further minor differences in the pro4b). The ‘M, 32 000 protein’ of Pleurodefes perties of the M,. 30000 protein(s) in difwaftlii also did not form direct immuno- ferent species were revealed by two-dimenprecipitates when reacted with the anti- sional gel electrophoresis (fig. 4) in which bodies against the M, 30000 protein of this protein showed a highly characteristic Xenopus but the antigen-antibody com- distribution. While it is markedly acidic in plexes formed could be precipitated by oocyte nuclei of all the species examined binding their Fc-portions to protein A of and appeared in the form of typical Staphylococcus aureus (fig. 3, slots 9 and ‘streaks’, representing groups of isoelectric Exp CellRes 129(1980)

Localization of a major soluble nuclear protein

Fig. 6. Indirect immunofluorescence microscopy of frozen section through a vitellogenic oocyte of Xenopus faevis seen in phase contrast (a) and in epifluorescence (b) optics after staining with guinea pig antibodies against the M, 30000 protein, following the procedure described under (1 iii) in Material and Meth12-801805

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ods. Fluorescence is seen only in the nucleus of the oocyte. The staining is present throughout the whole nuclear interior and no preferential staining of nucleolar masses (NO; in a) and chromosomes is recognized. Bars, 100pm.

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variants (figs 4, 5; cf [25]), the pattern of the isoelectric variants was different in different amphibian species. For example, the M,. 30000 protein of Xenopus laevis and B&o bufo oocytes contained molecules ranging in apparent IEPs from 4.7 to 6.0, with two major ‘spot’ components of mean apparent IEPs of 4.8 and 5.3 whereas the corresponding protein present in oocyte nuclei of Pleurodeles waltlii was less acidic, covering an IEP range from 5.4 to 6.5, with three major spot components of apparent IEPs of 5.5, 5.8, and 6.25. The typical ‘streak-like’, relatively indistinct appearance of this protein on two-dimensional gel electrophoresis was, at least to a great extent, due to differences in the degree of phosphorylation. This was directly demonstrated by treatment of total nuclear proteins from Xenopus oocyte nuclei with alkaline phosphatase which resulted in the concentration of most of the Coomassieblue-stained ‘A4, 30000 protein’ in two major ‘spot’ components, one with an apparent A4, of 30000 and an IEP of 4.9 and the other with an apparent A4, of 29 500 and an apparent IEP of 5.3 (fig. 5a-c). Sometimes minor spot components of apparent IEPs of 5.0 and 5.4 were also observed in such experimentally dephosphorylated protein (fig. 5~). The guinea pig antibodies to the native, i.e. highly phosphorylated M,. 30000 protein, also precipitated the protein(s) treated with alkaline phosphatase (data not shown here; see [32]).

Localization microscopy cells

by immunojluorescence in diverse amphibian

The guinea pig antibodies to the nuclear oocytes which formed precipitable antigen-antibody complexes only with this protein (see above ‘M, 30000 protein’ from Xenopus

and [25]) were used for indirect immunofluorescence microscopy on frozen sections of various amphibian tissues and on cultured kidney epithelial cells from Xenopus laevis (XLKE cells). In oocytes of frozensectioned ovaries a positive reaction was only observed in the nucleus (fig. 6) and the antigen protein appeared to be spread throughout the whole nuclear interior. Nucleoli, chromosomes and nuclear envelope did not show any special decoration [cf 251. The exclusively nuclear reaction was seen with whole antiserum as well as with purified immunoglobulin (IgG) and monospecific antibodies obtained from the purified antigen-IgG complexes by dissociation in high salt (fig. 6; for details of preparation see [25]). Nuclear staining was also obtained when the antibodies were applied in Tris-buffer containing 100 mM MgCl, (fig. 6), illustrating the high stability of the localization of the antibody complexes of the soluble M, 30000 protein (for discussion of the special problems and requirements for immunolocalization of soluble proteins see below). The antibodies against the M, 30000 protein from Xenopus laevis oocyte nuclei also showed strong and specific nuclear decoration in a variety of other cells in frozen sections of tissues of Xenopus and in interphase stages of cultured Xenopus (XLKE) cells (figs 7, 8). Cells showing positive nuclear staining included follicle epithelial cells of ovary [cf 251, hepatocytes, endothelial cells, and fibroblasts of liver [25], smooth and cross-striated muscle cells (for data see [32]), and interstitial cells, fibroblasts, Sertoli cells, spermatogonia, spermatocytes of testis (fig. 8). When incubation and washing periods were kept short (see below) the immunospecific fluorescence was usually dispersed throughout the nuclear interior, often revealing finely

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Fig. 7. Immunofluorescence microscopy showing kidney epithelial cells of Xenopus laevis (XLKE; cf [27]) grown in culture stained with the antibodies against the M, 30000 protein (a: phase contrast, b: epifluorescence optics) prepared according to the procedure

(3 ii) described in Methods. The nuclei show intensive fluorescence (b, inset in b) whereas no staining is detected in the cytoplasm. During mitosis the antigen is uniformly distributed in the cell (arrows in a and b denote the same mitotic cell). Bars, 20 pm.

granular depositions, and was not signilicantly enriched in nucleoli or aggregates of condensed chromatin (figs 7, 8). In mitotic stages, however, the staining was more disperse (fig. 7, inset), often spread over the whole cytoplasm. The same localization exclusive to the interior of nuclei was observed when the antibodies to the it4,. 30000 protein of Xenopus were used on frozen sections of various tissues of other amphibian species, including livers of Rana pipiens (fig. 9), R. esculenta (not shown here; see [32]), Z’riturus cristatus (not shown here; cf [32]) and Pleurodeles waltlii ([32]; cf fig. 11a) and ovaries of all the species listed in Materials and

Methods (for extensive documentation of observations see ref. [32]). The results, together with the immunoprecipitation data presented above, showed that’s broad range of oocytes and other cells of various, probably all, amphibia contain a protein which is similar to, and immunologically crossreacts with, the M, 30000 protein that is the most frequent protein of the oocyte nucleus of Xenopus laevis. Two types of cells were found which did not show reaction with the antibodies to the M,. 30000 nuclear protein of Xenopus, neither in the nucleus nor in the cytoplasm. These were the erythrocytes present in liver and muscle tissue of Xenopus laevis Exp CellRes 129 (1980)

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[cf 251, Rana pipiens (fig. 9), Triturus cris- parallel that were not fixed with formaldehyde but were incubated for the same tatus (not shown here) and Pleurodeles waltlii (not shown here) and the late sper- time (10 min) or longer with antibody solumatids and maturing spermatozoa of tions showed generally reduced and often Xenopus laevis (fig. 8). As a control, the ac- false-specific nucleolar fluorescence (e.g., cessibility of the internal proteins present in fig. 11b). A similar ‘pseudospecific’ perithe erythrocyte nuclei was demonstrated by nucleolar staining in cultured cells of using Xenopus (XLKE cells, fig. 11c) was seen immunofluorescence microscopy rabbit antibodies to bovine histone H2B when unfixed cells were incubated for 45 (fig. lo), and the accessibility of the sperm min with antibodies against the M, 30000 heads was shown using guinea pig anti- protein. With both cells or air-dried secbodies against an unidentified sperm head tions, fixation with formaldehyde, inclusion protein (kindly provided by G. Heil, this in- of relatively high amounts of divalent stitution). cations (e.g., 10-100 mM MgCl,) in the incubation buffers, and short incubation times Technical remarks on problems of (less than 10 min) proved to be favourable localization of ‘soluble’ proteins conditions for intense and faithful localizaby immunofuorescence tion of this soluble nuclear protein. microscopy

In the course of these experiments we learned that the localization of highly soluble antigens such as the M, 30000 protein by indirect immunofluorescence microscopy was different with different procedures and that routine incubation procedures originally developed for demonstrations of stably structure-bound proteins might lead to artifacts. For example, when frozen sections through liver of amphibians (Pleurodeles, fig. 11a, b) were prefixed with 2% formaldehyde and the incubation with antibodies against the M, 30000 protein was kept short (5-10 min) an overall, strong, and disperse nuclear fluorescence was visible (fig. 1la). Sections made in

Fig. 8. Immunofluorescence microscopy on frozen section of Xenopus laevis testes showing two seminiferous tubules (77 in uhase contrast (a) and fluorescence (b) optics after-incubation with the antibodies against the M, 30000 protein (staining procedure: 2, in Methods). The nucleiof interstitial c&, Sertoli cells, spermatogonia and spermatocytes are stained whereas later spermatids (e.g., in the right tubule) and maturing sperm ceils (left tubule) are not decorated by these antibodies. Bars, 20 pm.

Cross-reaction and localization by immunojluorescence microscopy on diverse cells of higher (avian and mammalian) vertebrates

The antibodies to the M, 30000 protein from Xenopus oocyte nuclei showed a strong and specific decoration of nuclei present in various tissues of chicken, including hepatocytes, tibroblasts and endothelial cells of frozen-sectioned liver (fig. 12). In this species also the nuclei of late stages of erythropoiesis, notably mature erythrocytes, were negative (fig. 12). Frozen sections through chick ovaries showed strong fluorescence in nuclei of follicular and thecal cells as well as in the oocyte nuclei (fig. 13). ‘Punctate’ granular distribution in the nuclei was commonly observed; the oocyte nuclei often showed increased fluorescence in the periphery (figs 12, 13). Embryonal chicken fibroblasts grown in culture also gave a positive, nucleus-specific reaction (not shown here). Similar results as in amphibian and avian cells were obtained in cultured mammalian Exp Cell Res 129 (1980)

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Exp CeNRes 129 (1980)

Localization

Fia. 10. Frozen section of Rana biens

liver in immunofluorescence microscopy, showing hepatocytes and a blood vessel (V) filled with erythrocytes (a: phase contrast; b: fluorescence optics). After incuba-

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tion with antibodies to histone H2B (procedure Ii of Methods) the nuclei of both cell types, hepatocytes and erythrocytes, are strongly stained. Bars, 20 pm.

cells from various species. For example, of it, and chromosomes showed greatly positive, specific nuclear staining was ob- reduced, if any staining (figs 14, 15). In all served when the antibodies to the soluble these cells it was important to keep the M,. 30000 protein from Xenopus oocyte nu- times of incubation and washing as short as clei were examined on cells from a mar- possible (see above). supial, i.e. rat kangaroo (fig. 14), murine In all tissues and cultured cells studied 3T3 cells (not shown here; for data see the antigen recognized also was found ex[32]), and on human HeLa cells (fig. 15). tractable during prolonged incubation and Again, the nuclear staining usually ap- washes with PBS, indicating its soluble peared in a punctate pattern and was not nature. specifically localized to nuclear substructures identifiable at the light microscopic level (figs 14, 15). During mitosis the antiDISCUSSION gen seemed to be spread over the cytoIn this and the preceding publication [25] plasm, at least in the more central portions we have described guinea pig antibodies directed against the most frequent nuclear protein present in oocytes of Xenopus Fig. 9. Frozen section through liver of Rana pipiens incubated with the M, 30000 antibodies according laevis. This is a soluble protein characprocedure 2 and seen in indirect immunofluorescence terized by its unusual resistance to heatmicroscopy (a: phase contrast; 6: epifluorescence). The nuclei of hepatocytes and endothelial cells (some induced denaturation, its high negative are denoted by arrows) show strong fluorescence, charge, its high degree of phosphorylation, whereas nucleated erythrocytes (in the blood vessel, V) show no reaction. Bars, 20 pm. and its complexity which shows a broad Exp Cell Res 129 (1980)

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Fig. II.

Immunofluorescence microsconv on frozen sections through liver of the newt, Pleuroheles waltlii (a. b) and cultured cells of the frog, Xenopus laevis (XLKE cells, c) after incubation with antibodies against the soluble M, 30000 protein. The micrographs illustrate the effects of different fixation and incubation procedures (a: procedure 2 as described in Methods; b, procedure 1ii as described in Methods; c: procedure 3i). While cells prefixed with formaldehyde (a) show an overall or at least broadly dispersed nuclear fluorescence, cells not fixed with formaldehyde and incubated for long periods of time (e.g., 10 min in b and 45 min in c) show only a ‘pseudospecific’ nucleolar or perinucleolar fluorescence due to elution of most of the antigen from other nuclear regions during the incubation and washing steps. Bars, 20 pm.

fers see [25]) are centrifuged at 120000 g for 1 h almost all of the protein is recovered in the supematant whereas no significant amounts are associated with the washed pellets which still contain intact-looking nucleoli as well as chromosome and nuclear envelope structures [25]. From such fractionation, the localization in situ, and the immunoprecipitations which have not shown any other ‘co-precipitated’ proteins we conclude that this protein is readily soluble and is diffusible in situ and in vivo. Thus, it falls into the category of nucleusbound soluble proteins but unlike other proteins of this category [cf 21, 221 it does not seem to be stably bound to nuclear structures in large amounts. The antibodies described are highly specific and react with the whole class of isoelectric variants [25] as well as with protein experimentally depleted in phosphate groups by treatment with alkaline phosphatase (see above and [32]). They also react with similar, if not identical protein(s) present in unfertilized eggs and various somatic cells of Xenopus as well as in nuclei of oocytes and many somatic cells of other amphibian species. The cross-reaction, however, is not limited to amphibia but is also found with an immunologically related 12. Immunofluorescence microscopy of frozen section of chicken liver showing hepatocytes and a larger blood vessel (V), which contains many erythrocytes (a: phase contrast, b: epifluorescence optics), after incubation with antibodies against the M, 30000 protein ofXenopus (preparation procedure 1ii). Nuclei of hepatocytes, Iibroblasts and endothelial cells are stained but no fluorescence is seen in nuclei of erythrocytes. Bars, 20 pm.

Fig.

range of isoelectric variants and at least two polypeptide components of slightly different molecular weights of approx. 30000 and 29500. In oocytes, the protein has only been detected in significant amounts in the Exp Cell Res 129 (1980)

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terization of the protein present in avian and mammalian cells will be presented elsewhere; these authors). The reason for these differences is not clear. While phosphorylation clearly contributes to the electronegative character of the protein(s) our experiments with enzymatic dephosphorylation and quantitative determinations of phosphorus contents indicate that the differences observed, e.g., between Xenopus and Pleurodeles, not only result from differences in phosphorylation (compare also, e.g., the data in figs 4b and 5~). Our data also show that the soluble acidic A4, 30000 protein is the most frequent nuclear protein in oocytes not only 0fXenopus laevis but also of all other amphibian species examined (see also [32]). For quantitative recovery of this protein in isolated nuclei, fast isolation (total time for isolation as described here: 30-60 set) and/or the presence of 5-10 mM MgCl* in the isolation Fig. 13. Immunofluorescence microscopy on frozen buffer is critical. During prolonged incubasection through chicken ovary, showing an early oocyte, that has been stained with antibodies against tions as well as in nuclei isolated in mass the M, 30000 nuclear protein from Xenopus laevis oocytes (preparation procedure liii as described in preparations (e.g., the procedure in ref. Methods). The oocyte nucleus (arrows denote the nu- 1401) a large proportion of this protein is clear periphery) as well as nuclei of follicle and thecal cells show intense and specific fluorescence. Bar, lost, together with a number of other dif50 pm. fusible proteins in germinal vesicles (see also the data in ref. [39]). In intact, freshly isolated nuclei of full-grown oocytes of protein present in nuclei of oocytes and Xenopus laevis the acidic M, 30 000 protein somatic cells of a representative of the birds represents 9-10% of the total nuclear pro(chick) and in a broad range of cells of mam- tein, i.e. 0.25-0.28 pg per nucleus, equimalian species, including human cells. This valent to about 6x 1012molecules per nusuggests that immunologically identical or cleus, i.e. ca 4.2 mg/ml, assuming a mean closely related nuclear protein(s) are of nuclear diameter of 500 ,um [cf 251. Hence, widespread occurrence among vertebrate this protein exceeds even the two other cacells and have been considerably stable tegories of unusually frequent proteins throughout vertebrate evolution. On the present in these oocyte nuclei, i.e. actin other hand, we have observed differences representing ca 8 % of nuclear protein, i.e. in electrophoretic mobility and the number 0.22 pg per nucleus, equivalent to ca 3 X 10” of negatively charged groups in this protein molecules per nucleus (i.e. ca 3.2 mglml; as even among difterent amphibian species to contents of nuclear actin in oocytes see (see above; detailed biochemical charac- also [7, 31, 38, 39]), and the histones (using Exp Cell Res 129(1980)

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Fig. 14. Immunofluorescence microscopy of rat kangaroo cells (PtK,) stained (procedure 3ii as in Methods) with antibodies to the nuclear M, 30000 protein fromxenopus laevis oocytes (a: phase contrast, b: epifluorescence optics). In interphase cells (e.g., the two cells in the center) antigen protein is located exclu-

sively in the nucleus, during mitosis (a metaphase is seen in the lower right, early and late telophase cells are shown in the left and in the inset) the antigen is spread over the cytoplasm but is absent in the chromosomes (e.g., inset in b). Bars, 20 pm.

a value of 50 ng per nucleus, cf [21,41]; i.e. about 1.2~ 1012molecules or ca 0.8 mglml, assuming a mean M, value of 25000). Similarly high concentrations of the acidic M, 30000 protein are present in oocyte nuclei of the other amphibian species examined. We do not know the absolute concentrations of this or the immunologically related protein(s) in the other, somatic cells;

however, the immunofluorescence microscopy suggests that it is also a relatively frequent protein. Currently, we are attempting to quantitatively determine the amount of the protein by immunoprecipitation from whole cell lysates. We are also examining whether the observed absence of the protein in nuclei of some transcriptionally inactive cells such as sperm cells

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Fig. 15. Immunofluorescence microscopy of cultured human cells (HeLa) incubated (according procedure 3ii) with antibodies against the nuclear M, 30000 protein of Xenopus laevis oocytes. In interphase, fluorescence is restricted to the nucleus whereas in mitosis (a telophase is shown in the lower left, an

Exp CellRes 129 (1980)

anaphase-to-telophase stage is seen in the lower right) antigen is also seen in the cytoplasm. In late telophase (lower left) fluorescence is also found disperse in the cytoplasm, but the antigen protein seems to concentrate in the nucleus. Bars, 20 pm.

Localization

and erythrocytes reflects the complete absence of the protein in these cells or masking by other nuclear components. Although our results allow the conclusion that an immunologically related soluble protein is present in nuclei of the diverse cell types and species examined, they do not per se demonstrate the identity of the protein in the different cells. As we show here, the acidic protein recognized by the antibodies displays some minor differences such as in apparent IEP and electrophoretic mobility even between oocytes of different amphibian species. On the other hand, the observed differences in electrophoretic mobility on sodium dodecylsulfate (SDS)polyacrylamide gels does not necessarily reflect differences in molecular weight but might well be due to some amino acid substitutions changing the character of the protein complexes with SDS or to differences in protein modification. The acidic M, 30000 protein described here is clearly different from other similar-sized nuclear molecules such as histones, including those of the Hl family, HGM (high mobility group) proteins (for data of this protein class see [43-46]; for localization see, however, also [47]), and the M, 31000 protein ‘BA’ described by Catino et al. [cf 481. We cannot fully exclude at the moment that it is related to the ‘protein 52’ described by Conner & Comings [49] to have an apparent M, of 25 000 although it appears to be less firmly retained in nuclei isolated and washed in various buffers and is of somewhat lower molecular weight (apparent M,, 25000). The extractability and localization characteristics of the acidic soluble protein discussed here are somewhat similar to those of the protein factor S-II that stimulates RNA polymerase II [50] but the latter is a basic protein and has an apparent M, of ca 40000 [51]. On the other hand, the M,

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30000 acidic nuclear protein localized in the present study appears to be identical with or closely related to the ‘nucleosome assembly factor’ protein of M, 29000 which Laskey and colleagues have prepared from Xenopus laevis egg homogenates [52, 53; see also 541. Recently, this group has further shown that the purified assembly factor protein from egg homogenates has a similar mobility and typical ‘streak-like’ appearance on two-dimensional gel electrophoresis as the nuclear protein described here (Mills, A D, Laskey, R A, Black, P & De Robertis, E M, personal communication). Moreover, these authors have also observed that this protein is rapidly translocated into the nucleus when injected into the ooplasm (same authors, personal communication), in agreement with the predominantly, if not exclusively nuclear location described by immunofluorescence microscopy in the present study. The function of this abundant protein in oocyte nuclei is not clear. Its high concentration suggests that it may serve a fundamental structural role rather than special catalytic processes. It could be that high concentrations of this protein are involved in charge neutralization of the excess of histones that has been described in the nuclei of amphibian oocytes [41, 531, but may also occur in other cells, at least in certain metabolic situations [e.g., 551. While this is possible, our finding that the M, 30000 protein can be immunoprecipitated from nuclear material solubilized in buffers of nearly physiological strength without histones co-precipitating (this study and ref. [25]) seems to argue against the existence of a considerable portion of this protein in stable complexes with histones. The experiments of Laskey and his colleagues [52-541 as well as the recent observations of Stein et al. [56] on effects of poly-(glutamic acid) have Exp Cell Res 129 (1980)

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provided a basis for the hypothesis that enucleated Xenopus laevis oocytes (not such acidic nuclear protein facilitates the shown here; for data see [32]), and the assembly of histone-histone complexes and significance of this low figure is hard to asnucleosomes. Similarly, acidic proteins sess. Likewise, the protein is only detected may also modify the stability and confu- in nuclear location in various interphase mation of histone-containing structures. cells. Therefore, we conclude that this proChromatin material is not replicated in vi- tein is a nuclear protein in the sense that it and full-grown amphibian is greatly enriched in the nucleoplasm (see tellogenic oocytes: however, the accumulated and also above). However, in contrast to other stabilized histone stored in these oocytes nuclear proteins such as histones which are may contribute to chromatin formation dur- associated with the chromosomal material ing early embryogenesis. On the other during mitosis (for an example shown by hand, we would draw attention to the pos- immunofluorescence microscopy see, e.g., sibility that such acidic proteins may also [lo]) this protein is spread over the cytofacilitate the formation of ribonucleoproplasm during mitosis (this study) and during teins which are known to be produced in meiotic division of amphibian oocytes (see large quantities in oocytes as well as in the also the data from the unfertilized eggs of other cell types in which this type of pro- Xenopus in refs [52, 531). Interestingly, the tein has been detected (this study). In the protein appears to be effectively re-achigh concentration present such acidic pro- cumulated within the confinements of the teins may provide an ‘environment’ protein reformed nuclear envelope during telophase facilitating certain nuclear functions or (this study). simply represent a specific nuclear ‘spaceIt is widely assumed that proteins that are filling’ sol-phase colloid. Our failure to de- rapidly and highly accumulated in the nutect protein(s) cross-reacting with the cleus are kept in this location by binding to soluble acidic M,. 30 000 protein of Xenopus other specific nuclear components [22, 591. in transcriptionally inactive nuclei charac- As to the most frequent nuclear protein of terized by a predominance of condensed the amphibian oocyte, the acidic MT 30000 chromatin also points to a possible correla- protein, we have not found evidence of the tion of this protein type with transcriptional existence of a considerable portion of this activity. Whether the protein is directly re- protein in a form complexed to larger struclated to transcriptional events or to nuclear tures or other proteins. On the other hand, and chromatin changes preceding transcrip- the data presented by Feldherr & Potional processes as, for example, demon- merantz [59] indicate that most of the M, 30000 protein soluble in vitro in living strated in reactivation of chick erythrocytes in heterokaryons [57, 581 remains to be ex- oocytes remains within nuclei even 2.5 h after experimentally puncturing the nuclear amined in future experiments. We have not been able to detect signi- envelope (see fig. 3 or ref. [59]). This perficant amounts of the M,. 30000 protein in plexing situation that a soluble protein readily extractable from isolated nuclei in the cytoplasm of oocytes by immunofluorescence microscopy (this study). Only as vitro is bound in vivo to nuclear structures little as 0.4% of the total oocyte content of in interphase and meiotic prophase nuclei but not during nuclear division awaits furthis protein has been immunoprecipitated from the solubilized ooplasmic material of ther experiments for an explanation. Exp CellRes 129 (1980)

Localization of a major soluble nuclear protein We thank Dr Marie-Christine Dabauvalle for help with the experiments involving alkaline phosphatase as well as Miss Stefanie Winter and Mr Klaus Mlhler (this institution) for excellent technical assistance. We are also indebted to Drs R. Laskey and E. M. De Robertis (MRC Laboratory of Molecular Biology, Cambridge, UK) for informing us about their unpublished tindings

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