Identification of the Major Nuclear Matrix Proteins

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Proc. NatI. Acad. Sci. USA Vol. 88, pp. 10312-10316, November 1991 Cell Biology Nuclear matrins: Identification of the major nuclear matrix proteins HIROSHI NAKAYASU* AND RONALD BEREZNEYt Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260 Communicated by Keith R. Porter, August 23, 1991 (received for review June 27, 1991)

ABSTRACT A preparative two-dimensional polyacrylam- MATERIALS AND METHODS ide gel system was used to separate and purify the major Isolation of Nuclei and Nuclear Matrix. Nuclei and nuclear Coomassie blue-stained proteins from the isolated rat liver matrix were isolated from rat liver by a modification of the nuclear matrix. Approximately 12 major proteins were con- high-salt extraction procedures of Berezney and Coffey (6, sistently found. Of these, 5 proteins represented identified 14, 15) as described (17). Phenylmethylsulfonyl fluoride (1 proteins, including nuclear lamins A, B, and C, the nucleolar mM) and sodium tetrathionate (0.1 mM) were added to all the protein B-23, and residual components of core heterogeneous isolation solutions through the high salt extraction (2 M NaCl) nuclear ribonucleoproteins. The remaining eight major pro- step (17). teins termed the nuclear matrins consisted of matrin 3 (125 Two-Dimensional (2D) Electrophoresis, Purification of In- kDa, slightly acidic), matrin 4 (105 kDa, basic), matrins D-G dividual Nuclear Matrix Proteins, and Preparation of Chicken (60-75 kDa, basic), and matrins 12 and 13 (42-48 kDa, acidic). Polyclonal Antibodies. The 2D nonequilibrium pH gradient Peptide mapping and two-dimensional immunoblot studies and SDS/PAGE system of O'Farrell et al. (18) was modified indicate that matrins D-G compose two pairs of related pro- to optimize separation of the major nuclear matrix proteins teins (matrins D/E and F/G) and that none of the matrins on a large scale. Total rat liver nuclear matrix protein (2-3 resemble the nuclear lamins or any of the other major proteins mg) was separated on 13-cm tube gels for the first- detected on our two-dimensional gels. Subfractionation immu- dimensional pH gradient (pH 3-10 ampholytes, 400 V, 14-16 noblot experiments demonstrated the nearly exclusive localiza- h, 22°C). The second-dimensional gels measured 15 cm x 30 tion of matrins F/G and other matrins to the nuclear matrix cm x 1.5 mm and consisted of a 2-cm 3.5% polyacrylamide fraction of the cell. These results were further supported by stacking gel on top of a 28-cm 5-12% gradient gel. Electro- indirect immunofluorescence microscopy that showed a strictly phoresis was performed at 15 V for 4 h followed by 250 V for interior nuclear localization of the matrins in intact cells in 20-24 h at 4°C. The gels were stained with Coomassie blue and the major stained spots were excised and stored at contrast to the peripherally located nuclear lamins. We con- -20°C. The individual proteins were further purified after clude that the nuclear matrins are a major class of proteins of extraction from the gel pieces (in 60 mM TrisHCl, pH the nuclear matrix interior and are distinct from the nuclear 6.8/0.1% SDS/2% 2-mercaptoethanol) on one-dimensional lamins. (1D) SDS/PAGE gels (10% polyacrylamide) according to Laemmli (19) and stored as gel slices at -20°C. There is a growing awareness that many of the important For polyclonal antibody production, multiple gel slices for answers to questions concerning the expression and regula- each protein were washed four times with 10 mM sodium tion of the eukaryotic genome will require an understanding phosphate, pH 7.2/0.15 M NaCl and homogenized in the of the higher-order arrangement and function of the genetic same buffer with 2 vol of Freund's complete or incomplete components as they interact within the complex three- adjuvant. Intramuscular and subcutaneous injections into dimensional architecture of the cell nucleus (1-13). The mature laying hens were given with complete adjuvant at day nuclear matrix, a residual nuclear structure that has been 1 (100-200 gg of protein) and every 2 weeks with incomplete isolated from a wide variety of eukaryotic cells throughout adjuvant (50-100 ,ug of protein). Sera were prepared at day the phylogenetic scale (1-3, 6, 7, 13-16), offers a potentially 28 and subsequently every 2 weeks. valuable in vitro approach for studying nuclear processes in 2D Western Blot Analysis. Total nuclear matrix protein (100 relation to nuclear structure. Indeed, numerous studies have ,ug) was separated on the 2D gel system described above on a for all a miniscale (5.5-cm tube gels and 5 cm x 7.5 cm x 0.45 mm implicated the matrix as site oforganization virtually slab gels) and electrophoretically transferred to a nitrocellu- known nuclear processes (1-11, 13, 16), such as, DNA loop lose sheet (20). After blocking with 0.2% Tween 20/10 mM attachment, DNA replication, transcription, RNA splicing Tris-HCI, pH 7.4/0.15 M NaCl for 2-4 h, the blots were and transport, hormone receptor function, carcinogen bind- stained with india ink (21) or incubated for 12 h with the ing, oncogene proteins, viral proteins, and protein phosphor- various polyclonal antibodies (1:100 dilution ofsera) followed ylation. by 2 h with alkaline phosphatase-conjugated goat anti- Despite this progress, our knowledge of the proteins that chicken IgG secondary antibodies (1:1000 dilution) and de- compose this proteinaceous nucleoskeletal structure is still in veloped as described (22). its infancy. In this study we have used high-resolution Peptide Analysis of Purified Nuclear Matrix Proteins. Pro- preparative PAGE to identify and purify many of the major teins were extracted from the gel slices as described above for Coomassie blue-stained nuclear matrix proteins. A class of the 2D gel pieces, precipitated with 3 vol of absolute ethanol nuclear matrix proteins termed the nuclear matrins is iden- (-20°C, 16 h), and dissolved in 0.1% SDS/60 mM Tris-HCI, tified and characterized by peptide maps, polyclonal anti- pH 6.8. For 1D peptide analysis, 3 1,u of each purified protein bodies generated against the individual purified matrins, and indirect immunofluorescence microscopy. Abbreviations: hnRNP, heterogeneous nuclear ribonucleoprotein; 1D and 2D, one and two dimensional, respectively. *Present address: Department of Pharmacology, Kyoto Prefectural The publication costs of this article were defrayed in part by page charge University of Medicine, Kawaramachi-Hirokoji, Kamikyo, Kyoto payment. This article must therefore be hereby marked "advertisement" 602, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 10312 Downloaded by guest on October 2, 2021 Cell Biology: Nakayasu and Berezney Proc. Natl. Acad. Sci. USA 88 (1991) 10313 solution (3 jig of protein) was incubated with 2 ,1l of either for certain experiments, and protein 1, which often appears L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated as a more minor protein by Coomassie blue staining. Since trypsin (80 ng; Sigma) or protease V8 (300 ng; Sigma) for 1 h our preparations include the nuclear lamins A, B, and C as at 370C and subjected to SDS/PAGE using either a 15% major components, we have also labeled a series of four (protease V8 digested) or 12% (trypsin digested) minigel (19). proteins, proteins D-G, that migrated at similar apparent The gels were stained with Coomassie blue. For 2D analysis, molecular masses (60-75 kDa) but are much more basic in 14 pug of each nuclear matrix protein was incubated with 160 charge. ng oftrypsin (370C, 1 h). The reactions were stopped with 0.1 The 2D pattern of the major Coomassie blue-stained poly- mM phenylmethylsulfonyl fluoride, and the peptides were peptides was extremely reproducible in rat liver and was also precipitated with ethanol and subjected to 2D SDS/PAGE characteristic of nuclear matrix isolated from a variety of using the 10%o polyacrylamide minigel system described other mammalian cells including HeLa S-3 (24), Chinese above. hamster lung cells (Don line), mouse 3T3 fibroblasts, and Affinity Purification of Chicken Polyclonal Antibodies and PtK1 rat kangaroo kidney cells (data not shown). Indirect Immunofluorescence Microscopy. For immunofluo- Relationship of Nuclear Matrix Proteins to Known Nuclear rescence microscopy, the sera containing the polyclonal Proteins. To determine whether any of the major proteins antibodies were first affinity purified on nitrocellulose. Nu- other than the nuclear lamins were known nuclear proteins, clear matrix proteins from 2D gels were transferred to we obtained a variety ofantibodies to nuclear proteins. These nitrocellulose (20). Individual spots corresponding to the included antibodies to core heterogeneous nuclear ribonu- major proteins were identified by brief staining with india ink cleoproteins (hnRNPs) A, B, and C (25-27), other presump- (21), excised, and incubated with the corresponding chicken tive hnRNPs (26, 27), proteins of the small nuclear RNP serum (16 h, room temperature). The filter spots were washed particles (28, 29), the nucleolar specific proteins B-23 or four times with 0.2% Tween 20/10 mM TrisHCl, pH 7.4/0.15 numatrin (30, 31), and C-23 or nucleolin (32, 33). Immunoblot M NaCI and distilled water. Affinity-purified antibodies were analysis showed that spot 14 is B-23 and spot 15 contains eluted from the nitrocellulose with 0.1 M glycine hydrochlo- hnRNP core proteins (data not shown). All the other anti- ride (pH 2.2) on ice for 5 min and immediately neutralized bodies to RNP and nucleolar proteins reacted only with more with 3 M Trizma base. Immunofluorescence microscopy was minor polypeptides on the 2D gels (data not shown). Proteins performed on rat kangaroo kidney (PtK1) cells grown on 3, 4, 12, 13, and D-G are, therefore, uncharacterized major coverslips as described (23). proteins of the nuclear matrix. We have termed these proteins the nuclear matrins to distinguish them from nuclear lamins A, B, and C. RESULTS Purification and Peptide Mapping of Individual Nuclear 2D Polyacrylamide Gel Electrophoresis and Identification of Matrix Proteins. We next purified large quantities of individ- Nuclear Matrix Proteins. Nuclear matrix proteins from rat ual major matrix proteins from 64 preparative 2D gels (Fig. liver were separated on a preparative 2D SDS/PAGE system 2c). The 1D V8 peptide maps of most of the proteins were (18). Whereas >50 spots were detected on the 2D gels (Fig. very different (Fig. 2a). As reported (34), the peptide maps 1), only =12 major Coomassie blue-stained proteins were of lamins A and C were very similar to each other but quite present. Most of these major nuclear matrix proteins mi- distinct from that of lamin B. Since matrins D-G were very grated between 60 and 200 kDa with an approximately equal resistant to V8 protease, we used trypsin digestion. The distribution between the acidic and basic sides ofthe gel. We digestion patterns of matrins D and E resembled each other have numbered these major proteins from 1 to 15 including a more minor protein at position 2, which served as a control a kDa 3 4 F G DE A C B 12 13 14 15 Acidic Basic 97- 68- *_ -- ..... kDa - - 45- ..- 2 35- _ q S 24- - - 4 97- 1 18- 14- 68-II _*!(i1t*B) 6(D) 5(F) A BO * G_ _W -7(G) 57- 6(E) . 12 1t(C) 45- 13 A B C D E F G A B C D E F G '4 68- __ (B-23) 15 45- (hnRNP) 35- 24- 18- FIG. 1. Preparative 2D PAGE of nuclear matrix proteins. Total 14- nuclear matrix proteins from rat liver were separated on a preparative (2-3 mg of protein) PAGE system. The major Coomassie blue-stained proteins are numerically labeled including one minor b c spot (protein 2) and another spot (protein 1) that often stain less intensely than shown. Nuclear lamins A, B, and C are indicated with FIG. 2. 1D peptide maps of nuclear matrix proteins. Nuclear letters along with a group of matrin proteins that migrate in the same matrix proteins purified from 2D gels (identified by lane labels) were molecular weight range (matrins D-G). A broken line indicates the digested with V8 protease (a) or trypsin (b) and subjected to 1D approximate position of pH 7 on the pH gradient. Molecular mass PAGE. (c) Purified proteins before protease digestion. Molecular markers from top to bottom were thyroglobulin, phosphorylase b, mass markers were, from top to bottom, bovine serum albumin, bovine serum albumin, pyruvate kinase, ovalbumin, and lactate ovalbumin, glyceraldehyde 3-phosphate, trypsinogen, lactoglobulin, dehydrogenase. and lysozyme. Downloaded by guest on October 2, 2021 10314 Cell Biology: Nakayasu and Berezney Proc. Natl. Acad Sci. USA 88 (1991) of lamins A and C (Fig. a but were very different from those 4 b 2b). Matrin G showed a digestion pattern distinct from the B A D F lamins and matrins D and E. * a C E G The 2D peptide maps of lamins A and C were very similar with a few differences indicated in the areas of the gels enclosed by broken lines (Fig. 3 a and b). Lamin B had a very different peptide pattern (results not shown). The 2D peptide maps of the matrin pairs D/E (Fig. 3 c and d) and F/G (Fig. C d 3 e and f), respectively, were also similar but very different from either the lamins or each other. *- (A,C) AP- (AC) Characterization of Nuclear Matrins with Polyclonal Anti- bodies. Polyclonal antibodies were raised in chickens to the purified major matrix proteins. Positive sera were obtained for matrins 3, 4, 13, and A-G. A high degree ofspecificity was indicated on 2D Western blots. Anti-matrin 3 and 4 antibodies e f *r (CE) reacted only with matrins 3 and 4, respectively (Fig. 4 i and (0, E) j). Both anti-lamin A and C antibodies reacted with both lamins A and C but not with lamin B (Fig. 4 c and d). This is consistent with the near identity oflamins A and C in primary structure (35-37). A similar cross reactivity was found with antibodies to matrins D and E (Fig. 4 e and]) and matrins F and G (Fig. 4 g and h), respectively. Antibodies to these 9 h proteins, however, did not react with lamins A, B, or C or -I (F, G) ' (FG) other matrix proteins. Our immunological results suggest a relationship between the protein pairs lamins A/C, matrins D/E, and matrins F/G and are consistent with the 2D peptide map studies of Fig. 3, which suggested significant similarity in the primary sequence of these pairs of proteins. The i (3) chicken sera raised against matrin 13 showed a relatively i '(4~) weak signal on Western blots. It did, however, cross react with matrin 12 (data not shown) suggesting that proteins 12 and 13 may represent another example of a related matrin pair. Cellular Distribution of the Nuclear Matrins. The subcellular distribution of the nuclear matrins in rat liver tissue was FIG. 4. 2D Western blots of nuclear matrix proteins. (a) then examined on 1D immunoblots (Fig. 5). Matrins F/G Coomassie blue-stained proteins. (b) Preimmune serum control. (c) were detected in isolated nuclei (lane 7), enriched in the Anti-lamin A. (d) Anti-lamin C. (e) Anti-matrin D. (f) Anti-matrin E. nuclear matrix fraction (lane 11), and depleted in the high salt (g) Anti-matrin F. (h) Anti-matrin G. (i) Anti-matrin 3. (j) Anti- extract (lane 9), which contains the bulk chromatin and most matrin 4. of the nuclear protein. Matrin F/G was not detected in any of the other subcellular fractions. The absence of a visible signal in the total liver homogenate fraction was anticipated since nuclear proteins are <5% ofthe total protein ofthe liver pH Gradient tissue. Our findings suggest an exclusive nuclear localization A - - BA 1 of matrins F/G in the cell and their virtually complete a b association with the nuclear matrix subnuclear fraction. Similar results were obtained for matrins D, E, 3, and 4 (data not shown). We next examined the structural localization ofthe nuclear matrins in whole cells by indirect immunofluorescence microscopy. Unlike lamins A and C, which are located along the ~laminA C nuclear periphery (Fig. 6a), the nuclear matrins are found e d Lamin exclusively within the nuclear interior with no significant staining of the nucleoli (Fig. 6 c, e, g, and i). The staining iC patterns for all the nuclear matrins examined appear identical and consist of a tightly packed network of fibrogranular structures.

DISCUSSION Nuclear matrix proteins are the nonhistone proteins found in the nuclear matrix subfraction after nuclease, salt, and de- tergent extraction of isolated cell nuclei (1-6, 13-16). Virtu- ally all known nuclear functions are associated with this proteinaceous nucleoskeletal structure (1-13, 16). Morpho- logically, the nuclear matrix consists of residual components FIG. 3. 2D peptide maps of nuclear matrix proteins. Nuclear of the nucleolus, nuclear envelope (generally termed the lamins A and C and matrins D-G were subjected to 2D peptide nuclear lamina), and an elaborate fibrogranular network that mapping after trypsin digestion. Peptides unique to a particular extends throughout the nuclear interior and is often termed protein are enclosed in a broken line. the internal matrix (1-6, 14-16). Progress has been made in Downloaded by guest on October 2, 2021 Cell Biology: Nakayasu and Berezney Proc. NatL. Acad. Sci. USA 88 (1991) 10315

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FIG. 5. Subcellular distribution ofmatrins F/G in rat liver tissue. A rat liver homogenate was centrifuged at 640 x g for 10 min (nuclei), 10,000 x g for 10 min (mitochondria), and 100,000 x g for 2 h (microsomes and cytosol). Equal amounts of protein from each fractiorl were analyzed on iD Western blots. (a) Nitrocellulose blot. Lanes: 1, whole homogenate; 2, postnuclear supernatant; 3, mitochondria; 4, postmitochondrial supernatant; 5, microsomes; 6, cytosol; 7, purified nuclei; 8, DNase supernatant; 9, high salt supernatant; 10, Triton X-100 supernatant; 11, nuclear matrix; 12, standard proteins, from top to bottom, 83-galactosidase, phosphorylase b, bovine serum albumin, ovalbumin, and carbonic anhydrase. (b) Corresponding Coomassie blue-stained gel. elucidating the three-dimensional structure of the matrix (13, 38, 39), and these results have led to a growing use ofisolated nuclear matrix as an in vitro system for studying the higher- order arrangement and expression of the eukaryotic genome (4-13, 16). Although our knowledge of the nuclear matrix proteins is very limited, it is clear that a detailed molecular analysis of the individual proteins is of paramount importance for deci- phering the structural organization and molecular details of FIG. 6. Immunofluorescence staining of fixed cells by nuclear matrin antibodies. PtK1 cells were grown on coverslips and fixed the functional processes associated with this intriguing nu- with freshly depolymerized 3% (wt/vol) parafortnaldehyde, and cleoskeletal structure. Previous studies using 1D SDS/PAGE immunofluorescence microscopy was performed with the anti- (2, 6), although useful for providing an initial indication ofthe matrin polyclonal antibodies. (a) Anti-lamin A/C. (c) Anti-matrin 3. overall polypeptide profile of the nuclear matrix, are ex- (e) Anti-matrin 4. (g) Anti-matrin D/E. (i) Anti-matrin F/G. (k) tremely limited due to the enormous complexity of the Preimmune serum control. (b, d, f, h, j, and 1) Corresponding protein composition. phase-contrast micrographs. (Bars = 4 jum.) The 2D analyses of nuclear matrix proteins performed by lines L. and several groups stress the high degree of complexity of these (ref. 24; H.N., Buchholtz, R.B., unpublished polypeptide profiles (13, 40-46). By using [35S]methionine data). labeling for detection, Fey and Penman (42) have detected Antibodies to known nuclear proteins revealed that five of >200 proteins in the nuclear matrix. Stuurman et al. (46) have the major Coomassie blue-stained proteins correspond to also found enormous complexity in the 2D profiles with the lamins A, B, and C, B-23 or numatrin, and hnRNP core sensitive silver staining procedure. Despite this complexity, proteins. The remaining eight proteins (termed nuclear ma- these studies have provided valuable information. For ex- trins to distinguish them from the nuclear lamins) consisted ample, the total nuclear matrix proteins can be separated into of matrins 3, 4, D-G, 12, and 13. Matrin 3, a high molecular two major classes: those found in a variety of cell lines weight slightly acidic protein of 125 kDa, likely does not (common matrix proteins) and those that are cell-type and correspond to the nuclear matrix protein "mitotin," which differentiation-state dependent (13, 42, 46, 47). migrates to a similar position on 2D gels but is found only in In this study we have developed a 2D PAGE system that proliferating cells (48). Matrin 4 (105 kDa, basic) likely optimally separates many of the major nuclear matrix pro- corresponds to p107, the nuclear-matrix-associated protein teins. We detected in rat liver nuclear matrix =12 major described by Smith et al. (49). Matrins 12 and 13 show no Coomassie blue-stained protein spots along with >50 more obvious relation to identified nuclear-matrix-associated pro- minor spots. A virtually identical pattern ofmajor Coomassie teins. A possible relationship to the 36- and 40-kDa nuclear blue-stained proteins was detected in 2D PAGE profiles of matrix proteins reported by Lehner et al. (50) remains to be nuclear matrix obtained from a variety of mammalian cell examined. Matrins D-G likely correspond to an ill-defined Downloaded by guest on October 2, 2021 10316 Cell Biology: Nakayasu and Berezney Proc. Natl. Acad Sci. USA 88 (1991) cluster of 60- to 75-kDa basic nuclear matrix proteins ob- 15. Berezney, R. & Coffey, D. S. (1977) J. Cell Biol. 73, 616-637. served in several studies (41, 43, 45, 46). Halikowski and 16. Agutter, P. S. & Richardson, J. C. W. (1980) J. Cell Sci. 44, Liew (51) have also identified a presumptive chromatin 395-435. protein(s) termed B2 that migrated in the same general 17. Basler, J. B., Hastie, N. D., Pietris, D., Matsui, S., Sandberg, position as this basic cluster. B2, which is also a highly A. & Berezney, R. (1981) Biochemistry 20, 6921-6929. 18. O'Farrell, P. Z., Goodman, H. M. & O'Farrell, P. H. (1977) phosphorylated protein, was shown to be also associated Cell 12, 1133-1142. with the nuclear matrix (52). 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Peptide mapping and antibody cross-reaction studies indi- 20. Burnett, W. N. (1981) Anal. Biochem. 112, 195-203. cated that the nuclear matrins are distinct from the nuclear 21. Hancock, K. & Tsang, V. C. (1983) Anal. Biochem. 133, lamins. Immunofluorescence microscopy, furthermore, re- 157-162. vealed a strictly interior nuclear location for the matrins as 22. Blake, M. S., Johnston, K. H., Russell-Johns, G. J. & Got- opposed to the peripherally located lamins. These results are schlich, E. C. (1984) Anal. Biochem. 136, 175-179. consistent with Kaufmann and Shaper (45) who reported that 23. Belgrader, P., Dey, R. & Berezney, R. (1991) J. Biol. Chem. components in the cluster of 60- to 75-kDa nuclear matrix 266, 9893-9899. proteins had 1D peptide maps different from the lamins and did 24. Belgrader, P., Siegel, A. J. & Berezney, R. (1991) J. Cell Sci. 98, 281-291. not recognize anti-lamin antibodies on immunoblots. Our 25. Kesser, G. P., Escarla-Wilke, J. & Martin, T. (1984) J. Biol. results further indicate that the four proteins identified in this Chem. 259, 1827-1833. cluster form two pairs of related proteins (matrins D/E and 26. Dreyfuss, G. (1986) Adv. Cell Biol. 2, 459-498. F/G). Matrins 12 and 13 may form a third pair and matrins 3 27. Dreyfuss, G., Choi, Y. D. & Adam, S. A. (1986) Mol. Cell. and 4 showed no relationship to any of the other matrix Biol. 4, 1104-1114. proteins. 28. Pettersson, I., Hinterburger, M., Mimori, T., Gottlieb, E. & We propose that the nuclear matrins compose a broad Steitz, J. (1984) J. Biol. Chem. 259, 5907-5914. family of structural proteins in the nucleus with potential 29. Billings, P. B., Allen, R. W., Jensen, F. C. & Hoch, S. O. subfamilies indicated by the various protein pair homologues. (1982) J. Immunol. 128, 1176-1180. 30. Feurstein, N., Spiegel, S. & Mond, J. J. (1988) J. Cell Biol. 107, The actual relationship of each putative protein pair, how- 1629-1642. ever, will require more detailed molecular studies. In this 31. Feurstein, N., Chan, P. K. & Mond, J. J. (1988) J. Biol. Chem. regard, this laboratory has demonstrated that matrins D/E 263, 10608-10612. and F/G specifically bind DNA on 2D Southwestern blots, 32. Olson, M. 0. J., Wallace, M. O., Herrera, A. M., Marshall- whereas matins 3, 12, and 13 do not (53). Moreover, the Carlson, L. & Hunt, R. C. (1986) Biochemistry 25, 484-491. cDNA coding regions for matrins F/G and 3 have been 33. Lapeyre, B., Bourbon, H. & Amalric, F. (1987) Proc. Natl. sequenced and are-as predicted from this study- Acad. Sci. USA 84, 1472-1476. completely unrelated (23, 54). Consistent with their DNA 34. Kaufmann, S. H., Gibson, W. & Shaper, J. H. (1983) J. Biol. the matrin structure con- Chem. 258, 2710-2719. binding properties, F/G primary 35. Laiberte, J.-F., Dagenais, A., Filion, M., Bibor-Hardy, V., tains putative zinc-finger DNA binding motifs (54), but no Simard, R. & Royal, A. (1984) J. Cell Biol. 98, 980-985. known DNA binding motif was identified in matrin 3 (P. 36. McKeon, F. D., Kirschner, M. N. & Caput, D. (1986) Nature Belgrader and R.B., unpublished data). (London) 319, 463-468. We thank the following investigators for their generous gifts of 37. Fisher, D. Z., Chandhary, N. & Blobel, G. (1986) Proc. Natl. antibodies: Terrence Martin for antibodies to hnRNP core proteins Acad. Sci. USA 83, 6450-6454. (iD2), small nuclear RNP proteins (SmY12), and the 70-kDa U1 small 38. Fey, E. G., Krochmalnic, G. & Penman, S. (1986) J. Cell Biol. nuclear RNP protein (2.73); Gideon Dreyfuss for antibodies to 102, 1654-1665. hnRNP C1 and C2 core proteins (2B12) and a 120-kDa hnRNP protein 39. He, D., Nickerson, J. A. & Penman, S. (1990) J. Cell Biol. 110, (3G6); Mark Olson for antibodies to B-23 (numatrin) and C-23 569-580. to Linda A. Buchholtz and 40. Peters, K. E., Okada, T. A. & Comings, D. E. (1982) Eur. J. (nucleolin). We are extremely grateful Biochem. 129, 221-232. Steven Rosenbloom for their expert technical assistance. Jim Stamos & provided the illustrations. This work was supported by National 41. Verheijen, R., Kuijpers, H., Vooijs, P., van Venrooij, W. Institutes of Health Grant GM-23922 Ramaekers, F. (1986) J. Cell Sci. 86, 173-190. (to R.B.). 42. Fey, E. G. & Penman, S. (1988) Proc. Natl. Acad. Sci. USA 85, 1. Berezney, R. & Coffey, D. S. (1976) Adv. Enzyme Regul. 14, 121-125. 63-100. 43. Peters, K. E. & Comings, D. E. (1980) J. CellBiol. 86, 135-155. 2. Berezney, R. (1979) in The Cell Nucleus, ed. Busch, H. 44. Milavetz, B. I. & Edwards, D. R. (1986) J. Cell. Physiol. 127, (Academic, New York), Vol. 7, pp. 413-456. 388-395. 3. Shaper, J. H., Pardoll, D. M., Kaufmann, S. H., Barrack, 45. Kaufmann, S. H. & Shaper, J. H. (1984) Exp. Cell Res. 155, E. R., Vogelstein, B. & Coffey, D. S. (1979) Adv. Enzyme 477-495. Regul. 17, 213-248. 46. Stuurman, N., Meijne, A. M. L., van der Pol, A. J., de Jong, 4. Berezney, R. (1985) UCLA Symp. Mol. Cell. Biol. 26, 99-117. L., van Driel, R. & van Renswoude, J. (1990) J. Biol. Chem. 5. Berezney, R. (1991) J. Cell. Biochem. 47, 109-124. 265, 5460-5465. 6. Berezney, R. (1984) in Chromosomal Nonhistone Proteins, ed. 47. Dworetzky, S. I., Fey, E. G., Penman, S., Lian, J. B., Stein, Hnilica, L. S. (CRC, Boca Raton, FL), Vol. 4, pp. 119-180. J. L. & Stein, G. (1990) Proc. Natl. Acad. Sci. USA 87, 7. Bouteille, M., Bouvier, D. & Seve, A. P. (1983) Int. Rev. Cytol. 4605-4609. 83, 135-182. 48. Philipova, R. N., Zhelev, N. Z., Todorov, I. T. & Hadjiolov, 8. Jackson, D. A. (1986) Trends Biochem. Sci. 11, 249-252. A. A. (1987) Biol. Cell 60, 1-8. 9. Gasser, S. M. & Laemmli, U. K. (1987) Trends Genetics 3, 49. Smith, H. C., Spector, D. L., Woodcock, L. F., Ochs, R. L. & 16-22. Bhojee, J. (1985) J. Cell Biol. 101, 560-567. 10. Bodnar, J. (1988) J. Theor. Biol. 132, 479-507. 50. Lehner, C. F., Eppenberger, H. M., Fakan, S. & Nigg, E. A. 11. Nelson, W. G., Pienta, K. J., Barrack, E. R. & Coffey, D. S. (1986) Exp. Cell Res. 162, 205-219. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 457-475. 51. Halikowski, M. J. & Liew, C. C. (1985) Biochem. J. 225, 12. Nigg, E. (1988) Int. Rev. Cytol. 110, 27-92. 357-363. 13. Nickerson, J. A., He, D., Fey, E. G. & Penman, S. (1990) in 52. Halikowski, M. J. & Liew, C. C. (1987) Biochem. J. 225, The Eukaryotic Nucleus: Molecular Biochemistry and Macro- 693-697. molecular Assemblies, eds. Strauss, P. R. & Wilson, S. H. 53. Hakes, D. J. & Berezney, R. (1991) J. Biol. Chem. 266, (Telford, Caldwell, NJ), Vol. 2, pp. 763-782. 11131-11140. 14. Berezney, R. & Coffey, D. S. (1974) Biochem. Biophys. Res. 54. Hakes, D. J. & Berezney, R. (1991) Proc. Natl. Acad. Sci. USA Commun. 60, 1410-1417. 88, 6186-6190. Downloaded by guest on October 2, 2021