Review

Ascribing functions to the lamin B receptor

DURGADAS P KASBEKAR Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India (Fax, 91-40- 7171195; Email, kas @ ccmb.ap.nic, in).

This article reviews the research on the inner nuclear membrane lamin B receptor (LBR). It focuses on the biochemical and immunological evidence for an LBR; the cloning of chicken, rat and human LBR cDNAs and genomic sequences; the lamin B-, chromatin-, DNA- and NLS-binding properties of the N-terminal domain and its phosphorylation by different kinases; the sterol C-14 reductase activity of the C-terminal domain; the use of yeast two-hybrid screens and co-immunoprecipitation to identify interacting ; and the probing of nuclear assembly and disassembly in living cells with LBR-GFP fusion proteins. The article concludes by considering a scenario whereby LBR levels might even regulate expression.

1. Introduction arise from the same gene by alternative RNA splicing (Lin and Worman 1993); whereas there are two different lamin The lamin B receptor (LBR) is an integral protein of the B proteins in somatic mammalian cells', and inner nuclear membrane of metazoan cells. In the litera- , that are encoded by different . During ture it is also referred to by its acronym LBR, and by its mitosis the A- and C-lamins become soluble but the B- apparent molecular weight in SDS-PAGE, p58. However lamins remain associated with the intracellular membrane not all usages of p58 refer to the LBR, (e.g., Ferrini et al vesicles that are thought to be the remnants of the nuclear 1994). The first paper on LBR appeared a little over ten membranes. This suggested that a receptor might couple the years ago (Worman et al 1988). Today, LBR is recog- B-lamins to the inner nuclear membrane. The nuclear nized as possibly playing a central role in the mitosis- lamina is a discontinuous structure and where it is disrupted related disassembly and reassembly of the nuclear the chromatin appears to be directly associated with the envelope (for a review see Gant and Wilson 1997). This inner nuclear membrane. It turns out that LBR can bind to article will focus on the discovery of diverse properties of chromatin as well, thus LBR is both well positioned LBR and conclude by speculating on whether LBR might and functionally equipped to mediate interactions at the also regulate . chromatin-lamina-membrane interface of the nucleus. The nuclear envelope is comprised of two membranes; the outer and inner nuclear membranes. The space between the two is called the perinuclear space. Nume- 2. The biochemical evidence for an LBR rous transcisternal connections are formed between these two membranes by the nuclear pores. The outer nuclear Avian erythrocytes are the tissue of choice for the membrane is continuous with the endoplasmic reticulum isolation of nuclear membrane proteins because they (ER) and, like the ER, it is studded with ribosomes on its contain nuclei but few other intracellular membranes. cytoplasmic surface. The perinuclear space is thus conti- Worman et al (1988) extracted a turkey erythrocyte nuous with the ER lumen. The inner nuclear membrane is nuclear envelope preparation with 8 M urea to remove the lined on its nucleoplasmic side by a filamentous mesh- peripherally bound lamins and used the lamin-depleted work, the nuclear lamina, whose major constituents are nuclear envelopes in binding assays with purified [1251]- the intermediate filament proteins called lamins. There labelled lamin B. The nuclear envelopes bound the are essentially two kinds of lamins; the A- and C-lamins labelled lamin B in a saturable and specific manner. This are neutral and the B-lamins are acidic. Lamins A and C binding was significantly greater than that of purified

Keywords. Nuclear envelope; sterol biosynthesis; Neurospora; membrane- interaction

J. Biosci., 24, No. 3, September 1999, pp 401-408. © Indian Academy of Sciences 401 402 Durgadas P Kasbekar

[lESI]-Iabelled lamin A and it was competitively inhibited with LBR. Phosphorylation by this kinase affects the by unlabelled lamin B but not by unlabelled lamin A. The association of LBR with other nuclear proteins. plasma membrane did not show specific binding with labelled lamin B. Scatchard analysis revealed that labelled 3. The immunological evidence for an LBR lamin B bound to the iamin-depleted nuclear envelopes with a Kd of approximately 0.2 gM and the LBR concen- Human patients with autoimmune disorders often contain tration was approximately 50% of the envelope protein. in their sera antibodies to nuclear antigens. Primary The labelled lamin B bound to a 58 kDa protein (p58) biliary cirrhosis (PBC) is a chronic autoimmune disease in Western blots of the urea-extracted nuclear envelope of the liver and is characterized by progressive infla- proteins. This protein was absent from the urea- and matory destruction of the intrahepatic bile ducts with alkaline-extracts of the nuclear envelopes but it could be eventual progression to cirrhosis. A subset of patients solubilized by a mixture of 2% Triton X-100 and 2 M with PBC were found to harbour antibodies to turkey KC1. This showed that p58 is an integral membrane erythrocyte LBR (Courvalin et al 1990). This implied that protein. Antibodies raised against purified p58 blocked the autoantigen, human LBR, is conserved between birds the binding of labelled lamin B to the lamin-depleted and mammals. Subsequently Lassoued et al (1991) used nuclear envelopes and thereby confirmed that p58 and ELISA to show that the anti-LBR autoantibodies inhibited LBR are indeed the same protein. Indirect immuno- the binding to lamins of anti-lamin B autoantibodies but fluorescence microscopy with the anti-p58 antibodies did not affect the binding to lamins of anti-lamin A and C showed a nuclear rim staining indicating that p58 is antibodies. This provided additional evidence that LBR is primarily localized in the inner nuclear membrane. The indeed a specific receptor for lamin B and suggested that presence of a membrane receptor for lamin B provided an anti-LBR antibodies bear the internal image of the explanation for why lamin B unlike lamins A and C binding site of lamin B to LBR. remains membrane bound during mitosis. Incubation of turkey erythrocytes with 3ZPi followed by 4. Cloning of LBR cDNAs and genomic sequences analysis of the proteins in the urea extracted nuclear envelopes by SDS-PAGE and autoradiography revealed a Degenerate oligonucleotides were designed based on the 58 kDa phosphoprotein that comigrated with the p58 N-terminal amino acid microsequence of purified turkey antigen in a parallel immunoblot (Appelbaum et al 1990). erythrocyte LBR and used to perform RT-PCR with This indicated that p58 is phosphorylated in vivo. To chicken liver poly A enriched RNA (Worman et al 1990). examine whether dephosphorylation of p58 affected its An amplified product of the expected size was obtained binding to lamin B, nuclear envelope proteins were and the amino acid sequence derived from it matched that separated by SDS-PAGE and transferred to replica of the turkey derived microsequence. A synthetic oligo- nitrocellulose membranes. One membrane was dephos- nucleotide corresponding to a portion of the sense strand phorylated in situ with calf intestinal alkaline phosphatase of the PCR product was used to screen a chicken liver and the other was mock treated and then both were cDNA library. Overlapping cDNA clones were obtained incubated with [lasI]-lamin B. The p58 in the alkaline and their nucleotide sequences were determined to obtain phosphatase treated filter bound less labelled lamin B the primary structure of chicken LBR. The human cDNA relative to the mock treated filter. To verify that this clones were subsequently isolated by probing a HeLa cell reduction was not due to proteolysis of p58 during cDNA library with the chicken LBR cDNA at reduced incubation with alkaline phosphatase the p58 bands were stringency (Ye and Worman 1994). The chicken and eluted from the filters, re-electrophoresed in a gel and human cDNAs encoded proteins of 637 and 615 amino immunoblotted with anti-p58 antibodies. acid residues, respectively, that shared 68% amino acid To further explore the regulation of the membrane- sequence identity throughout their entire length. lamina interaction by phosphorylation, Courvalin et al The human cDNA was used to isolate genomic clones (1992) immunoprecipitated in vivo labelled LBR from to determine the structure of the human LBR gene interphase and mitosis-arrested chicken cells. They found (Schuler et al 1994). This gene spans 35 kb and has 13 that LBR was phosphorylated only on Ser residues in coding exons. Fluorescence in situ hybridization (FISH) interphase cells whereas in mitotic cells it was localized the LBR gene to cytogenetic location 1q42.1 phosphorylated on Ser and Thr residues, and some Ser (Wydner et al 1996). This location is close to FRA1H a residues phosphorylated in interphase were not phos- common 5-azacytidine fragile site. phorylated in mitosis. Additionally LBR was shown to be a substrate for a p34cecE-type mitosi s specific kinase. This 5. LBR is also a nuclear localization signal strengthened the hypothesis that mitotic phosphorylation binding protein of LBR might contribute to the dissociation of the lamina from the inner nuclear membrane. A subsequent section The cDNA for rat LBR was cloned somewhat seren- describes an LBR kinase that was co-immunoprecipitated dipitously by a group studying nuclear envelope proteins LBR function 403

that could bind to nuclear localization signals (NLS). PBC sera, and nuclear rim staining with these antibodies Haino et al (1993) resolved a rat liver nuclear extract by was confirmed by immunofluorescence microscopy (Ye SDS-PAGE and ligand blotted it with [125I]-labelled and Worman 1994). The fusion proteins were also used to nucleoplasmin, a protein with a strong NLS. A 60 kDa concentrate rat lamin B from a dilute solution. In other protein was detected and designated NBP60 (for NLS experiments the fusion proteins were shown to retard the binding protein 60 kDa). NBP60 also bound to the NLS electrophoretic migration of double stranded M13 DNA of SV40 large T-antigen (T-peptide) conjugated to human in agarose gels. South-western blot analysis of fusion serum albumin. The binding of NBP60 to nucleoplasmin- proteins containing different stretches of the NTD with sepharose was specific because it was inhibited by the [32p]-labelled ~. DNA revealed that the stretch between wild-type T-peptide and not by a mutant T-peptide in Pro 2 and Ala 100 is required for DNA binding. which an essential Lys residue was replaced with Thr. As mentioned earlier rat LBR was identified in a search NBP60 could be extracted with 2% Triton X-100, 1 M for NLS binding proteins. To verify that human LBR also KCI but not with 1 M KCI-2 M urea or 2% Triton X-100 possesses NLS binding activity Kawahire et al (1997) and it partitioned into the lower layer in a 2-phase system tested GST-NTD fusion proteins for binding to labelled using Triton X-114. These results showed that NBP60 is T-peptide. In this way the NLS-binding activity was an integral membrane protein. NBP60 was purified by localized to residues 1-89 which contains the RS (Arg- nucleoplasmin-sepharose affinity chromatography and Ser) rich region. This binding was competitively inhibited hydroxyapatite HPLC and on the basis of its partial amino by unlabelled T-peptide but not by a mutant T-peptide. acid sequence PCR primers were designed to isolate The significance of LBR's NLS-binding activity remains clones from a cDNA library. The nucleotide sequence of to be determined. As described later the RS rich region is the cDNA revealed that NBP60 is a 620 amino acid the target for LBR kinase and for binding to the splicing protein with 79% and 63% identity to human and chicken factor 2 (SF2)-associated protein p34/p32. LBR (Kawahire et al 1997). In other words, NBP60 is rat LBR. 7. The LBR CTD is a sterol C-14 i'eductase

6. LBR is comprised of two structural domains with The LBR CTD cannot be studied in aqueous buffers because of its hydrophobic nature. However its homology different functions with fungal sterol C-14 reductases suggested that it might function as a sterol C-14 reductase. Prakash et al (1998, All three LBR proteins for which the deduced primary structure is known (chicken, rat and human) have two 1999) examined the CTD of human LBR for comple- mentation of the Neurospora crassa sterol C-14 reductase identifiable domains. The amino terminal one-third of the protein (approximately 200 amino acid residues) is mutant erg-3. Recombinant genes encoding proteins chi- meric for different amino acid sequences of Neurospora hydrophilic and basic whereas the carboxyl terminal two- sterol C-14 reductase and human LBR CTD were thirds (approximately 400 residues) is quite hydrophobic constructed and transformed into the erg-3 mutant strain. and contains 8 putative transmembrane segments. The All the chimeric constructs tested were able to com- amino terminal domain (NTD) contains consensus sites plement the biochanin A sensitive phenotype of the erg-3 for phosphorylation by protein kinase A, the mitosis specific kinase p34 cdc2, and a stretch that is rich in Arg- mutant (see figure 1) and UV spectrophotometry con- Ser dipeptides (RS). It also contains DNA binding motifs firmed that the transformants could synthesize ergosterol. This demonstrated that the LBR CTD is indeed a sterol that are common to several gene regulatory proteins and C-14 reductase. Independently, Silve et al (1998) showed histones. Remarkably, the carboxyl terminal domains (CTD) are highly similar (-40% amino acid sequence that human LBR can function as a sterol C-14 reductase in yeast. Novel questions emerge from these findings: Can identity) to the ergosterol biosynthetic enzyme sterol C-14 sterol C-14 reductase inhibitors interfere with LBR reductase of fungi and yeast (Papavinasasundaram and function? Are sterol changes involved in nuclear envelope Kasbekar 1994; Schuler et al 1994; Ye and Worman breakdown and reformation? 1994; D P Kasbekar, unpublished results). The hydrophilic nature of the LBR NTD made it a more convenient candidate (compared to the CTD) for study as 8. TM7SF2 and DHCR7 encode paralogues of the a recombinant fusion protein with glutathione S trans- LBR CTD ferase (GST). GST-NTD fusion proteins were expressed in Escherichia coli and were purified by affinity Recently two genes, TM7SF2 and DHCR7, were iden- chromatography on glutathione-sepharose followed by tified on human llq13 on the basis of the elution with reduced glutathione (Smith and Johnson similarity of their predicted proteins to the LBR CTD 1988). The human LBR NTD-GST fusion protein was (Holmer et al 1998). The DHCR7 protein contains 475 used to affinity purify the anti-LBR autoantibodies from amino acid residues and it has been shown to be the 404 Durgadas P Kasbekar

Figure 1. LBR-erg-3 chimeric genes can complement the biochanin A sensitivity phenotype of the N. crassa erg-3 mutant. Top panel: Vogel's-FGS agar plates were supplemented with biochanin A and streaked with conidia of transformants of the erg-3 mutant strain with three different LBR-erg-3 chimeric constructs (a-c); the wild type (d) and the untransformed erg-3 mutant (e). The plates were incubated at 30°C. Note that all three transformants and the wild type are resistant to biochanin A whereas the untransformed mutant is sensitive. The bar represents 333 p.m. Bottom panel: Schematic structures of the LBR-erg-3 chimeric proteins CH1, CH2 and CH3. The unstippled boxes represent the amino acid residues derived from erg-3 and the stippled boxes represent those derived from LBR. The numbers above the boxes refer to the residue numbers in the chimeric protein and the numbers below the boxes refer to the residues in LBR. LBR function 405 cholesterol biosynthetic enzyme 7-dehydrocholesterol chromo-shadow domains are separated by a hinge region reductase (Moebius et al 1998). Mutations in DHCR7 of- 70 amino acids. Experiments using the GST-HP1HS~' cause the Smith-Lemli-Opitz syndrome (Fitzky et al fusion proteins or the yeast two-hybrid assay showed that 1998; Wassif et al 1998; Waterham et al 1998), which is the chromo-shadow domain mediates the self associations the second most common autosomal recessive genetic of HPl-type proteins as well as their binding to LBR disorder (after cystic fibrosis) in the US white population NTD. (carrier frequency 1/200). The TM7SF2 protein is com- HCA of the LBR NTD also revealed two globular prised of 418 amino acid residues and its function is yet to domains separated by a hinge region. The first domain is be determined. The TM7SF2 protein shows about 40% between amino acid residues 1 and 60 and the second amino acid sequence identity with the Neurospora erg-3 between 105 and 210. The globular domains of the LBR protein and recent results from our laboratory showed NTD are distinct from the chromodomain and chromo- that a protein chimeric for TM7SF2 and erg-3 sequences shadow domains of HPl-type proteins. Yeast two-hybrid could complement the Neurospora erg-3 mutant assays and binding assays of in vitro translated fragments (A Prakash and D P Kasbekar, unpublished results). This of the LBR NTD to GST-HP1 ns'x fusion proteins showed suggests that TM7SF2 is also a sterol C-14 reductase. A that the stretch bearing residues 97-174, which contains a variant TM7SF2 protein of 590 amino acid residues was portion of the second globular domain, mediates the reported by Lemmens et al (1998). Unless a mistake has binding of LBR to HP1HS% Interestingly the Thr residue been made while sequencing, this variant might represent reported to be phosphorylated in mitosis (Courvalin et al a rare alternatively spliced transcript of TM7SF2. 1992) is in the second globular domain. It was suggested that this phosphorylation may disrupt the binding between LBR and the HPl-type proteins at the onset of mitosis 9. Identification of interacting proteins: when the inner nuclear membrane dissociates from the Yeast two-hybrid screens chromatin. And conversely, its dephosphorylation at the end of mitosis might function in the targeting of LBR- Ye and Worman (1996) cloned the cDNA for human LBR bearing membrane vesicles to the decondensing chromatin NTD into the GAL4 DNA binding domain fusion vector (Pyrpasopoulou et al 1996; Ye et al 1997). and co-transformed the resulting plasmid into yeast cells together with a library of ~ 106 recombinants of HeLa cell cDNA in the GAL4 activation domain fusion vector 10. Identification of interacting proteins: (pGADGH). Two co-transformants that showed the yeast Immunoprecipitation of the p58 complex two-hybrid interaction (Fields and Sternglanz 1994), were isolated and the inserts in their pGADGH-derived plas- To identify other factors that may modify or interact with mids were sequenced. One encoded the protein HP1Hs'~ p58 in vivo Simos and Georgatos (1992) subjected and the other encoded the majority of a novel protein with chicken erythrocyte nuclei to mild detergent extraction 65% sequence similarity to HP1HS% The latter protein and immunoisolated p58 with an affinity purified antibody was designated HP1Hs~. Both HP1 ns~ and HP1Hs~ are against a synthetic peptide comprised of the amino acid homologous to the Drosophila melanogaster hetero- residues 61-80 (R1 peptide) of the chicken LBR NTD. chromatin-associated protein HP1 which is a suppressor Coomassie brilliant blue staining of the SDS-PAGE of position effect variegation (James et al 1989; analysed immunoisolate showed up only the 58 kDa LBR Eissenberg et al 1990). The two HPl-like proteins were band but silver staining revealed additional minor bands synthesized in an in vitro transcription-translation system that were not present if the immunoprecipitation was done and shown to bind specifically to LBR NTD-GST coupled in the presence of excess R1 peptide. The appearance of glutathione-sepharose. The in vitro binding of LBR-NTD apparently specific co-immunoprecipitating bands sugges- to the HPl-like proteins confirmed the authenticity of the ted that at least a fraction of p58 is solubilized as a interactions detected in the yeast two-hybrid assay. multicomponent complex. The complex included the An algorithm called hydrophobic cluster analysis lamins A, B and C as shown by Western analysis and (HCA) was performed on the amino acid sequences of the additional proteins that migrated at 18, 34 and 150 kDa. HPl-type proteins and the LBR NTD (Ye et al 1997). The p34 could be eluted with 1 M NaC1 from the turkey This revealed that the HP1 proteins are comprised of p58 complex purified on an anti-R1 immunoaffinity two homologous but distinct globular domains. The N- column. It was purified further on a Sephacryl S100 terminal domain corresponded to the chromodomain column followed by SDS-PAGE, and subjected to originally identified on the basis of the homology between microsequencing following transfer to PVDF membrane Drosophila HP1 and Polycomb, a protein involved in and excision of the 34 kDa band. The sequence showed down-regulating homeotic genes during development that p34 is the avian homologue of the human nuclear (Paro and Hogness 1991). The C-terminal domain was protein p32 which is associated with splicing factor 2 designated the chromo-shadow domain. The chromo- and (SF2) (Simos and Georgatos 1994). This homology was 406 Durgadas P Kasbekar

confirmed by demonstrating that turkey p34 is recognized freely. Presumably the tight binding of the NTD to the by affinity purified antibodies raised against the N- and heterochromatin and/or lamina led to immobilization of C-terminal residues of HeLa p32. The p34/p32 may the LBR-GFP in the nuclear envelope. Thus changes in function as a linking component between the inner nuclear LBR-GFP mobility within the ER/nuclear envelope mem- membrane and structures involved in RNA splicing. Inte- brane system appear to be responsible for its localization restingly, SF2 contains an RS-rich region which is similar to the inner nuclear membrane during interphase. to the RS-rich region of the LBR NTD. The RS-rich During prometaphase the LBR-GFP redistributed from domains appear to provide the binding sites for p34/p32. the nuclear envelope into the interconnected ER and Incubation of the p58 complex with [~2p]ATP and then exhibited the same high mobility and diffusion as analysis by SDS-PAGE and autoradiography showed observed in the interphase ER membranes. This indicated heavy phosphorylation of the p58 band and indicated the that during nuclear envelope disassembly the nuclear presence of a specific LBR kinase in the complex. This membrane loses its nucleoplasmic contacts and becomes kinase was distinct from protein kinase A and cdc2 kinase 'resorbed' into the ER network. At the end of mitosis the because it was not inhibited by PKI, an inhibitor of ER membrane domains containing the LBR-GFP re- protein kinase A or by L1, a specific peptide inhibitor of establish contacts with the and wrap around cdc2. These inhibitors could inhibit the phosphorylation the chromatin as a newly formed nuclear envelope. of p58 by their cognate kinases. The LBR kinase migrated Antibodies to an LBR-like protein have been used to as a 110 kDa protein in SDS-PAGE. It was shown to examine the ordered recruitment of functionally discrete phosphorylate specific Ser residues in the RS region of vesicle types during nuclear envelope reassembly in the NTD of GST-NTD fusion proteins and in synthetic Xenopus (Drummond et al 1999). peptides representing different sequences from LBR In summary, these experiments revealed that nuclear (Nikolakaki et al 1996, 1997). Phosphorylation by the envelope disassembly occurs by dissolution of the nuclear LBR kinase reduced the binding of LBR to p34/p32. membranes into the surrounding ER network rather than p18 turned out to be an integral membrane protein that by fragmentation and vesiculation; and that nuclear is primarily expressed in erythrocytes but not in liver cells envelope reassembly involves ER reorganization around and which is distributed equally between the inner and decondensing chromatin. outer nuclear membranes (Simos et al 1996). It is similar to the mitochondrial isoquinoline-binding protein, p18 12. Might LBR regulate genes and differentiation? could also bind to B-lamins, but less strongly than to LBR. The p18 protein may be internally linked to the TM Overexpression of the LBR-GFP fusion protein in the domain of LBR. COS-7 cells (see § 11) resulted in the formation of nuclear membrane invaginations into the nucleoplasm (Ellenberg 11. LBR as a probe for nuclear envelope et al 1997). In contrast, Smith and Blobel (1994) had assembly and disassembly reported that in yeast cells expressing chicken LBR, membrane stacks were formed in locations that are typical Ellenberg et al (1997, 1998) expressed a fusion protein of of yeast ER. The membrane stacks presumably repre- LBR with green fluorescence protein (GFP) in living sented the additional ER generated by the yeast cells to COS-7 cells. The fusion protein contained the first 238 accommodate the heterologous LBR protein. The forma- amino acid residues of full length LBR followed by GFP. tion of membrane stacks in yeast but invaginations in Therefore it included the LBR NTD and first trans- COS-7 cells might reflect the "drawing in" of the extra membrane span which have been shown previously to be membrane into the nucleoplasm by the interaction sufficient to target heterologous proteins to the inner between the LBR NTD and HPl-type proteins in the nuclear membrane (Smith and Blobel 1993; Soullam and COS-7 cells. Yeast nuclei might not show this interaction Worman 1993, 1995). This segment contains the binding either because they do not contain as much hetero- domains for the three nucleoplasmic ligands of LBR; chromatin as the COS-7 cells or because they lack lamin B, HP1 and DNA. Confocal-time lapse imaging of proteins that interact with the heterologous LBR. the fusion protein in interphase cells showed that it was Extending this idea further, it is conceivable that LBR synthesized in the RER, from where it diffused to the levels might affect the extent to which the nuclear inner nuclear membrane. About 8 h after injection of the membrane and heterochromatin interact. plasmid construct there was a 5.3-fold higher level of Treatment of HL-60 human myeloid leukemia cells LBR-GFP fluorescence in the nuclear envelope relative with retinoic acid stimulates them to differentiate along to the ER. Fluorescence recovery after photobleaching the granulocyte pathway. Granulocyte differentiation (FRAP) analysis revealed that the LBR-GFP in the induces major ultrastructural changes of the nucleus nuclear envelope is immobile but the subpopulation that including the formation of lobules and production of remains in the ER is mobile and diffuses rapidly and envelope-limited chromatin sheets (ELCS) (Olins et al LBR function 407

1998). Concomitant with this increase in nuclear to its nuclear receptor is affected by phosphorylation; J. Biol. membrane and membrane-associated chromatin, there is a Chem. 265 4181-4184 Blobel G 1985 Gene gating: A hypothesis; Proc. Natl. Acad. significant increase in the amounts of LBR and two Sci. USA 82 8527-8529 antigenically related smaller peptides of 50 and 35 kDa. Collas P, Courvalin J C and Poccia D 1996 Targeting of Might the key element underlying these changes simply membranes to sea urchin sperm chromatin is mediated by a be a retinoic acid induced increase in LBR expression? lamin B receptor-like integral membrane protein; J. Cell Biol. Might this happen even during normal granulocyte 135 1715-1725 Courvalin J-C, Lassoued K, Worman H J and Blobel G 1990 differentiation? Identification and characterisation of autoantibodies against The hypothesis that the chromatin makes nonrandom the nuclear envelope lamin B receptor from patients with contacts with the inner nuclear surface has acquired primary biliary cirrhosis; J. Exp. Med. 172 961-967 increasing experimental support (Mathog et al 1984; Courvalin J-C, Segil N, Blobel G and Worman H J 1992 The Nagele et al 1999). The binding of LBR to HPl-type lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for a chromatin proteins is consistent with this hypothesis. p34CdcZ-type protein kinase; J. Biol. Chem. 267 19035-19038 Blobel (1985) had suggested that the nuclear context in Drummond S, Ferrigno P, Lyon C, Murphy J, Goldberg M, which genes operate is critical for their expression. If Allen T, Smythe C and Hutchison C J 1999 Temporal diffe- LBR levels can alter this context by changing the number rences in the appearance of NEP-B78 and an LBR-like of heterochromatin-nuclear membrane contacts it is protein during Xenopus nuclear envelope reassembly reflect the ordered recruitment of functionally discrete vesicle types; conceivable that it might affect the expression of genes J. Cell Biol. 144 225-240 susceptible to position effect variegation. One way to test Eissenberg J C, James T C, Foster-Hartnett D M, Hartnett T, this idea might be to sort the COS-7 cells on the basis of Ngan V, Elgin S C R 1990 Mutation in a heterochromatin- their LBR-GFP fluorescence intensity and search for specific chromosomal protein is associated with suppression transcript differences by differential display PCR. of position effect variegation in Drosophila melanogaster; Proc. Natl. Acad. Sci. USA 87 9923-9927 Ellenberg J, Siggia E D, Moreira J E, Smith C L Presley J F, Worman H J and Lippincott-Schwartz J 1997 Nuclear mem- 13. Conclusion brane dynamics and reassembly in living cells: Targeting of an inner nuclear membrane protein in interphase and mitosis; J. Cell Biol. 138 1193-1206 Studies with LBR have yielded new insights into the Ellenberg J, Lippincott-Schwartz J and Presley J F 1998 relationship between the nuclear envelope and the ER; I Two-color green fluorescent protein time-lapse imaging; they have provided a novel probe to study the dynamic Biotechniques 25 838-846 interactions between the chromatin, splicing machinery, Ferrini S, Cambiaggi A, Meazza R, Sforzini S, Marciano S, nucleoskeleton and intracellular membranes and hinted at Mingari M C and Moretta L 1994 T cell clones expressing the natural killer cell-related p58 receptor molecule display a role for sterol alterations in nuclear envelope assembly heterogeniety in phenotypic properties and p58 function; Eur. and disassembly. The discovery of genes coding for J. Immunol. 24 2294-2298 protein paralogues of the LBR CTD but which lack Fields S and Sternglanz R 1994 The two-hybrid system: an assay sequences equivalent to the LBR NTD has provided new for protein-protein interactions; Trends Genet. 8 286-292 and exciting prospects for studying gene evolution. So it Fitzky B U, Witsch-Baumgartner M, Erdel M, Lee J N, Paik Y K, Glossmann H, Utermann G and Moebius F F 1998 is not at all surprising that the LBR community has come Mutations in the delta7-sterol reduetase gene in patients with to "expect the unexpected". the Smith-Lemli-Opitz syndrome; Proc. Natl. Acad. Sci. USA 95 8181-8186 Gant T M and Wilson K L 1997 Nuclear assembly; Annu. Rev. Acknowledgements Cell Dev. Biol. 13 669-695 Haino M, Kawahire S, Omata S and Horigome T 1993 I thank J Gowrishankar for his careful reading of the Purification of a 60 kDa nuclear localization signal binding manuscript and useful suggestions, Vincent Colot and protein in rat liver nuclear envelopes and characterization of its properties; J. Biochem. 113 308-313 Howard Worman for encouragment, and A Prakash for Holmer L, Pezhman A and Worman H J 1998 The human lamin the figure. Work in my laboratory is supported in part by B receptor/sterol reductase multigene family; Genomics 54 the Departments of Biotechnology and of Science and 469-476 Technology, New Delhi, the Indo-French Centre for the James T C, Eissenberg J C, Craig C, Dietrich V, Hobsin A and Promotion of Advanced Research (Project 1403-2) and • Elgin S C R 1989 Distribution patterns of HP1, a hetero- chromatin-associated nonhistone chromosomal protein of the Third World Academy of Sciences (96-230 RG/ Drosophila; Eur. J. Cell Biol. 50 170-180 BIO/AS). Kawahire S, Takeuchi M, Gohshi T, Sasagawa S, Shimada M, Takahashi M, Abe T K, Ueda T, Kuwano R, Hikawa A, Ichimur/~ T, Omata S and Horigome T 1997 eDNA cloning of References nuclear localization signal binding protein NBP60, a rat homologue of lamin B receptor, and identification of binding Appelbaum J, Blobel G and Georgatos S D 1990 In vivo sites of human lamin B receptor for nuclear localization phosphorylation of the lamin B receptor. Binding of lamin B signals and chromatin; J. Biochem. 121 881-889 408 Durgadas P Kasbekar

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MS received 20 April 1999; accepted 19 July 1999

Corresponding editor: STUARTA NEWMAN