Using a solution binding assay, we show that purified 1251-labeled lamin B binds in a saturable and specific fashion to lamin-depleted avian erythrocyte nuclear membranes with a Kd of -0.2 ,LM. This binding is significantly greater than the binding of 12'I-labeled lamin A and is competitively inhibited by unlabeled ligand. We demonstrate that a 58-kDa integral membrane protein (p58) is a lamin B receptor by virtue of its abundance in the nuclear envelope and association with '251-labeled lamin B in ligand blotting assays. Specific antibodies raised against p58 recognize one protein in isolated nuclei and partially block 1251-labeled lamin B binding to lamin-depleted nuclear membranes. Cell fractionation and indirect immunofluorescence microscopy show that p58 is located in the periphery of the nucleus. This protein may serve as a membrane attachment site for the nuclear lamina by acting as a specific receptor for lamin B.The nuclear lamina is a filamentous meshwork situated between the inner nuclear membrane and heterochromatin (1-3). This structure provides support for the adjacent inner nuclear membrane (4) and possible attachment sites for chromatin (2, 5, 6) and cytoplasmic intermediate filaments (7). The nuclear lamins, a family of interrelated polypeptides structurally homologous to intermediate filament proteins, are the building blocks of the nuclear lamina (3,(8)(9)(10). Two major types of lamins have been recognized in higher eukaryotic cells: the acidic B-lamins and the neutral A-lamins (10-12). During mitosis and lamina depolymerization, the B-lamins remain associated with intracellular membrane vesicles thought to be remnants of the inner nuclear membrane, whereas the A-lamins become soluble (12). The B-lamins are also more resistant to extraction from the nuclear envelope compared to the A-lamins (13). Because of these findings, the B-lamins are thought to mediate the coupling of the lamina to the inner nuclear membrane (5, 13). It has been proposed that lamin B associates with the membrane either by a hydrophobic interaction with components of the lipid bilayer (14) or by an attachment to a transmembrane receptor protein (2, 13). These questions and controversies prompted the present investigations. MATERIALS AND METHODSCell Fractionation and Protein Purification. Nuclear envelopes and plasma membranes from turkey erythrocytes were prepared as previously described (7). Lamin-depleted envelopes were prepared by extraction with 8 M urea/10 mM Tris HCl, pH 8.0/1 mM EDTA (see below). Lamins A and B were purified from the urea extract of turkey or rat nuclear envelopes by DEAE-cellulose chromatography and were '25I-labeled with Bolton-Hunter reagent as described (7).Extractions with (i) 8 M urea/10 mM Tris HCl, pH 8.0/1 mM EDTA, (ii) 0.10 M Na2CO3 (pH 11.5), and (iii) 2% Triton X-100/2 M KCl/10 mM Tris-HCl, pH 8.0, were performed on nuclear envelopes at room temperature. All extraction solutions also contained 1 mM dithiothreitol and 0.2 mM phenylmethylsulfonyl fluoride. Bath sonication was use...

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylationdependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and crosslink these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.

Morphological studies have established that peripheral heterochromatin is closely associated with the nuclear envelope. The tight coupling of the two structures has been attributed to nuclear lamins and lamin‐associated proteins; however, it remains to be determined which of these elements are essential and which play an auxiliary role in nuclear envelope‐chromatin interactions. To address this question, we have used as a model system in vitro reconstituted vesicles assembled from octyl glucoside‐solubilized nuclear envelopes. Comparing the chromosome binding properties of normal, immunodepleted and chemically extracted vesicles, we have arrived at the conclusion that the principal chromatin anchorage site at the nuclear envelope is the lamin B receptor (LBR), a ubiquitous integral protein of the inner nuclear membrane. Consistent with this interpretation, purified LBR binds directly to chromatin fragments and decorates the surface of chromosomes in a distinctive banding pattern.

Previous studies have identified a subassembly of nuclear envelope proteins, termed "the LBR complex." This complex includes the lamin B receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32). The latter polypeptide has been shown to interact with the HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2). Using recombinant proteins produced in bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine-serine (RS) dipeptide motifs located at the nucleoplasmic, NH2-terminal domain of LBR and in members of the SR family of splicing factors. Furthermore, we show that the NH2-terminal domain of LBR binds to p34/p32, whereas a mutated domain lacking the RS region does not. Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex.

Using heterochromatin-enriched fractions, we have detected specific binding of mononucleosomes to the N-terminal domain of the inner nuclear membrane protein lamin B receptor. Mass spectrometric analysis reveals that LBR-associated particles contain complex patterns of methylated/acetylated histones and are devoid of "euchromatic" epigenetic marks. LBR binds heterochromatin as a higher oligomer and forms distinct nuclear envelope microdomains in vivo. The organization of these membrane assemblies is affected significantly in heterozygous ic (ichthyosis) mutants, resulting in a variety of structural abnormalities and nuclear defects.A significant proportion of heterochromatin is localized in the periphery of the cell nucleus and maintains a close spatial association with the inner nuclear membrane (1-5). This spatial association reflects a multiplicity of interactions between chromatin components and integral or peripheral proteins of the nuclear envelope (NE) 1 (6, 7).Because chromatin is extensively and differentially modified (8, 9), it is tempting to think that certain epigenetic marks or factors associated with histone modifying enzymes provide binding sites for NE proteins. However, it is equally possible that transcriptionally active, noncondensed chromatin is subjected to silencing and "heterochromatinization" upon contact to the NE. Both of these hypotheses receive experimental support: chromatin that is silenced through binding to SIRs can tether itself to the NE (10), whereas targeting of marker genes to the inner nuclear membrane suppresses their expression (11).One of the factors that have been implicated in chromatin anchorage to the NE is the lamin B receptor (12). LBR is a polytopic inner nuclear membrane protein consisting of a long, N-terminal domain, seven or eight hydrophobic transmembrane regions, and a C-terminal tail (13). The N-terminal part of the molecule protrudes to the nucleoplasm and contains multiple serine-arginine motifs that are phosphorylated by the SRPK1 and the cdc2 kinases (14, 15); the hydrophobic region represents, instead, a (functional) form of sterol reductase and is involved in cholesterol metabolism (16).Immunodepletion of LBR from detergent-solubilized NE vesicles results in proteoliposomes with a diminished ability to bind chromatin. Furthermore, direct binding of electrophoretically purified LBR to metaphase chromosomes can be demonstrated by in vitro assays (17). Corroborating these observations, anti-LBR antibodies block nuclear assembly in sea urchin egg extracts (18), whereas direct (19) and indirect (20) interactions with heterochromatin protein 1 (HP1) have been claimed in the literature.Two critical parameters in LBR-chromatin interactions are the physical state of LBR and the molecular features of LBRassociated chromatin. To address these issues, we have isolated fragments of peripheral heterochromatin attached to the inner nuclear membrane. These subcellular fractions were utilized to affinity select mononucleosomes that associate with LBR and investigate L...

Abstract. We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous ~25I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A+B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70 % of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.THOUGH an association between 10-nm filaments and the nuclear envelope has been proposed several times during the past on the basis of morphological observations (9, 14), evidence for such an interaction on the molecular level was obtained only recently. We have demonstrated in a preceding article (7) that vimentin subunits can bind under in vitro conditions to some proteinaceous receptor site displayed along the nuclear periphery. In this interaction vimentin uses its 6.6-kD carboxy-terminal tail domain to associate with the nuclear membrane and then polymerizes in situ, forming extensive filament arrays surrounding the nuclear envelope.Knowing that the vimentin receptor at the nuclear surface is of a proteinaceous nature and exploiting the in vitro binding assay established previously, we tried to identify such a filament attachment site. Pivotal criteria in this survey have been the following: (a) removal of the putative receptor from the envelopes by chemical extraction should in principle decrease the binding of mI-vimentin to the nuclear envelopes, (b) blockage of the same site by antibodies should affect the binding, and (c) the receptor protein should be able to bind vimentin in vitro and with the same site specificity as the previously described nuclear attachment site (7). Materials and Methods Extractions of Nuclear MembranesEqual amounts of turkey RBC nuclear envelopes (287 lag of protein) were washed once with 10 vol of 10 mM NaPO4, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) pH 7.6 at 4°C, pelleted, and resuspended in 0.5 ml of either (a) 20 mM NaPO4, 140 mM NaC1, 1 mM MgCI~, 0.1 mM PMSF, pH 7.6 (PBS+), (b) 1 mM Na2CO3, 1 mM EDTA, 2 mM 13-mercaptoethanol (13-me), 0.1 mM PMSF, pH 9.6, (c) 25 mM Tris-HCl, 140 mM NaCI, 1 mM EDTA, 2 mM I~-me, 0.1 mM PMSF, 1% Tween-20, pH 7.6, or (d) 10 mM NaPO4, 1 mM EDTA, 2 mM 13-me, 0.1 mM PMSE 1.5-6 M urea, pH 7.6. The s...

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1a, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were ef®ciently transported into the nucleus and targeted speci®c chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to puri®ed nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His 6 -tagged peptide representing this region inhibited recruitment of LAP2b and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.