©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cDNA Cloning and Characterization of Lamina-associated Polypeptide 1C (LAP1C), an Integral Protein of the Inner Nuclear Membrane (*)

Lori Martin , Cristina Crimaudo (§) , Larry Gerace (¶)

From the (1) Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lamina-associated polypeptides 1A-1C (LAPs1A-1C) are related integral membrane proteins of the inner nuclear membrane that bind to both A- and B-type lamins and have a putative role in the membrane attachment and assembly of the nuclear lamina. In this study, we have cloned a cDNA encoding LAP1C. The DNA sequence predicts a 506-amino acid protein of largely hydrophilic character with a single membrane-spanning region between residues 311-333. Mapping of the epitope recognized by the anti-LAP1 monoclonal antibody RL13 indicates that the hydrophilic domain containing residues 1-310 is exposed to the nucleoplasm and thus that LAP1C is a type II integral membrane protein. A second class of LAP1 cDNAs was isolated that contains two protein-coding nucleotide insertions in the LAP1C sequence. These probably encode parts of LAPs1A and/or -1B, suggesting that LAP1 isotypes arise from alternative splicing. Immunoblot analysis of mouse P19 teratocarcinoma cells and the P19MES-differentiated derivative of the latter suggest that LAP1 isotypes are differentially expressed during development, similar to members of the nuclear lamin family. Since the different LAP1 isotypes appear to bind lamins with different affinities, these changes in expression could be important for developmentally regulated alterations in nuclear structure.


INTRODUCTION

The nuclear envelope (NE),() the membrane boundary of the nucleus in eukaryotic cells, consists of inner and outer nuclear membranes, nuclear pore complexes, and nuclear lamina (1) . The lamina is a filamentous protein meshwork lining the inner nuclear membrane, which is thought to provide a structural framework for the NE and an anchoring site at the nuclear periphery for interphase chromosomes (2, 3, 4) . The lamina contains mainly a polymer of nuclear lamins, members of the intermediate filament protein superfamily (2, 3) . A number of less abundant polypeptides are associated with the lamina as well, including certain integral membrane proteins (see below).

Mammalian lamins have been classified into A and B subtypes, based on their sequence properties and state of membrane association during mitosis. B type lamins (lamins Band B) are present in somatic cells throughout development and remain membrane attached during M-phase, while A type lamins (lamins A and C) are expressed only during or following terminal differentiation in most cells and are not membrane-associated during mitosis (1, 3) . Recent studies have shown that both A- and B-type lamins bind to chromatin (5, 6, 7) or DNA (8, 9, 10) . Thus, it is likely that lamins play a direct role in the attachment of chromatin to the NE, although integral membrane proteins of the inner nuclear membrane also may contribute to this interaction (11) .

Newly synthesized lamins A, B, and Bare isoprenylated at carboxyl-terminal Caa X motifs, and this modification is important for their targeting to the inner nuclear membrane (2) . While the isoprenylated tail of lamin A is proteolytically removed after the latter is assembled in the NE, B-type lamins remain stably isoprenylated. Lamin-attached lipid is likely to promote the association of the lamina with the inner nuclear membrane, possibly by direct interaction with lipid bilayer. However, by analogy to other systems involving protein prenylation (12) , it is likely that integral membrane proteins also are important for the lamina-inner membrane interaction (13) . Recent studies have identified several integral membrane proteins localized to the inner nuclear membrane that bind to lamins in vitro, which could play a role in this lamina attachment. These include lamina-associated polypeptides (LAPs)1 and -2 of mammalian cells (14, 15) and a 54/58-kDa polypeptide found in avian and human cells (16, 17, 18, 19) that is homologous to sterol C14 reductase of Saccharomyces cerevisiae (4, 11) . Also, a 53-kDa protein (otefin) of the inner nuclear membrane of Drosphila cells had been described (20) , although its lamin binding has not been examined.

LAP2 is a 51-kDa polypeptide that specifically binds to both lamin B and chromatin in a mitotic phosphorylation-regulated manner (15) . cDNA cloning has shown that LAP2 contains a large hydrophilic amino-terminal domain exposed to the nucleoplasm and a single predicted membrane-spanning segment near its carboxyl terminus.() Three different isotypes of LAP1 ranging in size from 55-75 kDa (LAPs1A, -1B, and -1C) have been described in mammalian cells (14, 15) . In this study, we carried out cDNA cloning of LAP1C. We have found it to consist of 506 amino acids and have a substantially hydrophilic character, with only a single putative transmembrane segment. Monoclonal antibody epitope mapping combined with previous immunogold electron microscopy demonstrated that LAP1C is a type II integral membrane protein with approximately 60% of its mass exposed to the nucleoplasm. Analysis of LAP1 expression in liver and cultured cells suggested that substantial changes in the expression of LAP1 isotypes occur during development. Since different LAP1 isotypes appear to bind lamins with different affinities, this has important implications for understanding developmentally regulated changes in the organization of the nucleus.


EXPERIMENTAL PROCEDURES

Isolation of cDNA Clones for LAP1

RNA was isolated from rat livers (Sprague-Dawley males, 6-8 weeks) using the guanidinium isothiocyanate method (21) , and polyadenylated RNA was prepared by chromatography on an oligo(dT) cellulose column. The poly(A)RNA was used for preparation of a cDNA library in the LAPII expression vector (by Marjorie Snead, Stratagene) using a mixture of random and oligo(dT) primers. The cDNA library contained 3.3 10primary clones, with a background of 5% non-recombinants. Approximately 4 10primary clones were screened with RL13 (-LAP1) monoclonal antibody (14) that had been preadsorbed on an Escherichia coli/phage lysate. One positive clone with an insert size of approximately 2 kb (clone CO) was isolated. Oligonucleotides corresponding to the 5`-end of the clone CO were end labeled with T4 polynucleotide kinase (Promega) and used to probe a rat liver gt11 cDNA library from Clontech, yielding a full-length clone for LAP1C (clone CL). Clone CO differs from clone CL in that it contains two additional short insertions of coding sequence. The first is found after nucleotide 713 of clone CL and extends 81 nucleotides, encoding 27 novel amino acids. The second is found after nucleotide 1059 of clone CL and extends 151 nucleotides, resulting in loss of residue 335 of the CL sequence and addition of 51 novel amino acids.

In attempts to isolate additional CO-related clones, CO was labeled via the random-primed labeling method (Stratagene) and was used for nucleic acid screening of this same unamplified library, yielding three more independent clones. Six independent clones were found in a Stratagene rat hepatoma library, and three more were isolated from a rat macrophage library (generously provided by Dr. George Fey), all using this initial clone as a nucleotide probe. Restriction digestion and/or nucleotide sequencing indicated that seven of these clones contained either one or both of the insertions characteristic of CO, depending on whether they were sufficiently long enough to include both insertion sites. However, none of these clones extended sufficiently in the 5`-direction to yield cDNAs that would be predicted to encode the amino-terminal ends of LAP1A or 1B. The longest clone containing these two insertions had a 5`-end coinciding with nt 103 of LAP1C (predicted to begin at residue 17 of LAP1C).

The original CL clone lacked a guanine at nt 1264, compared with all other LAP1-related cDNAs that were isolated (with or without the CO-like insertions). This would result in a stop after amino acid 412 of LAP1C (Fig. 1). Since all three LAP1 isotypes contain the sequence predicted at residues 466-479 of LAP1C as shown by reaction with anti-peptide antibodies against this region, this nucleotide loss is very likely a cloning artifact, and a CL clone reconstructed to contain this missing nucleotide (Fig. 1) was used for this study.


Figure 1: DNA sequence and deduced amino acid sequence of LAP1C. The boxed amino acids comprise a predicted membrane-spanning region. The underlined amino acids were used to raise anti-peptide antibodies in guinea pigs. Arrowheads indicate the two sites where sequence insertions were found in multiple LAP1C-related clones (see Fig. 5). The Kyte and Doolittle hydrophobicity plot (31) shown at the bottom was generated using the GCG software package with an 11-amino acid window.



Plasmids were rescued from ZAPII clones according to the manufacturer's instructions; inserts were subcloned into pBluescript SK (Stratagene) from gt11 clones. All plasmids were propagated in XL-1 Blue cells (Stratagene). DNA was sequenced using the dideoxy chain termination method. Sequencing primers were synthesized as needed to obtain sequence information from both DNA strands. Sequences were analyzed using IntelliGenetics and GCG software.

Northern Blot Analysis

Total and poly(A)RNA from HTC cells and poly(A)RNA from rat liver were subjected to denaturing agarose gel electrophoresis (22) . After electroblotting onto HybondN transfer membrane (Amersham Corp.) in 25 m M sodium phosphate buffer at 600 mA for 1.5 h, the RNA was cross-linked to the membrane using ultraviolet light. The 5`- EcoRI fragment of clone CO (containing base pairs 417-1059 of LAP1C plus additional insertions; see above) was used to probe the blot. Hybridization was done in 5 SSPE (22) , 50% formamide, 0.1% SDS, 0.5% nonfat dry milk, and 100 µg/ml denatured, sonicated salmon sperm DNA at 37 °C. Filters were washed in 0.1 SSC (22) , 0.1% SDS at 55 °C.

Cell Culture

HTC, NIH 3T3, and P19 cells were from ATCC, and P19MES cells (23) were kindly provided by Brian Burke. HTC, NIH 3T3, and P19MES cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, and P19 cells were grown in -minimal essential Eagle's medium (Life Technologies, Inc.) containing 7.5% bovine serum and 2.5% fetal bovine serum.

In Vitro Translation and in Vivo Cell Labeling

In vitro transcription and translation was performed on both intact and linearized plasmid DNA using the Promega TNT-coupled transcription translation kit according to the manufacturer's instructions. For pulse labeling of cells in vivo, HTC cells grown to 75% confluency were rinsed in L-methionine- and L-cysteine-deficient Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% dialyzed fetal calf serum (starvation medium). Cells were then incubated in starvation medium for 30 min at 37 °C. Starvation medium containing 0.5 mCi/ml TranS label (ICN Biomedicals) was added to the cells for 30 min, and cells were immediately harvested as described for immunoprecipitation. For overnight labeling, cells were maintained in labeling medium supplemented with cysteine and 20% of the normal level of methionine with 50 µCi/ml TranS label.

Preparation of NEs

NEs were isolated from rat liver and were salt washed in a buffer containing 0.5 M NaCl as described (24) . To prepare a NE-enriched fraction from cultured cells, cell monolayers were scraped into cold phosphate-buffered saline (PBS) (10 m M sodium phosphate (pH 7.4), 140 m M NaCl) and were pelleted at 100 g for 5 min. Cells were then washed by resuspension and pelleting in cold homogenization buffer (HB) (10 m M Tris-HCl (pH 8.0), 10 m M KCl, 1.5 m M MgCl, 1 m M dithiothreitol, and protease inhibitors (1 m M phenylmethylsulfonyl fluoride and 1 µg/ml each of aprotinin, leupeptin, and pepstatin)) and were swelled in one packed cell volume of HB on ice for 20 min. Cells were then disrupted with 20-30 strokes in a tight fitting glass Dounce homogenizer. Nuclei were pelleted at 500 g for 5 min, resuspended in five packed cell volumes HB, given two more strokes in the Dounce homogenizer, and repelleted. Crude nuclear pellets were then brought to one cell volume of HB containing 50 µg/ml each of DNaseI and RNase A. After 20 min in a room temperature water bath, the digested nuclei were cooled to 0 °C. Next, an equal volume of 2 M NaCl, 10 m M Tris-HCl (pH 8.0), 1 m M dithiothreitol, and protease inhibitors was added, and NEs were immediately pelleted out of this solution at 2300 g for 10 min and frozen in liquid N.

Antibodies

Antibodies were raised in rabbits against a trpE fusion protein containing a portion of LAP1. The expression construct was made by ligating an EcoRI fragment from clone CO into the PATH1 vector (25) . The trpE fusion protein contained the LAP1C region extending from residues 120-334 plus two short additional sequences: the 27 amino acids of the first CO insertion (see above) and the first 5 amino acids of the second. The fusion protein was expressed in E. coli, purified by electrophoresis on an SDS gel, and used to immunize rabbits. To prepare an affinity matrix for purifying anti-LAP1 antibodies from the rabbit antiserum, the EcoRI fragment from clone CO (above) was subcloned into pGEX2T (Pharmacia Biotech Inc.). This second fusion protein was expressed, purified on glutathione-Sepharose, and coupled to CnBr-activated Sepharose (Pharmacia) at 0.5-1.0 mg/ml according to the manufacturer's instructions. Antibodies were adsorbed to the affinity column for 1 h at room temperature and, after washing of the column, were eluted with 3 M potassium thiocyanate, 0.5 M ammonium acetate, and 5 mg/ml bovine serum albumin (26) . Antibodies to a synthetic peptide corresponding to amino acids 466-479 of LAP1C were produced by coupling the peptide to keyhole limpet hemocyanin and injecting the conjugate into guinea pigs. Monoclonal RL13 IgG (14) was purified on protein G-Sepharose and coupled to CnBr-activated Sepharose at 3-5 mg/ml for use as an immunoadsorbent.

Immunoadsorption

Extracts of cultured cells for immunoadsorption were obtained by rinsing cells in PBS and scraping into 1% Triton X-100, 20 m M Tris-HCl (8.8), 500 m M NaCl, 2 m M EDTA, and protease inhibitors (solubilization buffer). The cell lysate was sonicated briefly, incubated on ice for 30 min, and spun at 265,000 g for 20 min to obtain a supernatant. Isolated rat liver NEs were solubilized in a similar manner, while in vitro translation products were simply diluted with 4 volumes of solubilization buffer. For immunoadsorption of LAP1 antigens from these various extracts, solubilized samples were incubated with RL13-Sepharose immunobeads overnight at 4 °C. Antibody beads were then washed four times with solubilization buffer and once with solubilization buffer lacking Triton X-100. Bound proteins were eluted in SDS sample buffer lacking dithiothreitol.

Gel Electrophoresis, Immunoblotting, and Immunofluorescence Microscopy

SDS-polyacrylamide gel electrophoresis was carried out on 10% polyacrylamide gels as described (27) , and immunoblotting was performed essentially as previously described (24) . RL13 was used at 2.5 µg/ml and was detected with peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories Inc.) diluted 1:1000. Affinity-purified antifusion protein antibody was detected with peroxidase-conjugated donkey anti-rabbit IgG 1:1000 (Jackson). Guinea pig anti-peptide serum against residues 466-479 of LAP1C was diluted 1:100. In competition experiments, the diluted antibody was preincubated with 0.5 m M immunizing peptide. Guinea pig antisera against lamins A/C and lamin B were used at 1:500 dilution. Peroxidase-conjugated donkey anti-guinea pig IgG 1:5000 (Jackson) was used to detect guinea pig antibodies. ECL reagents (Amersham Corp.) were used to detect all the secondary antibodies. For immunofluorescence microscopy, cells grown on coverslips were fixed for 4 min in 4% formaldehyde in PBS and were permeabilized for 4 min in 0.2% Triton X-100 in PBS. All antibodies were diluted in 0.2% gelatin-containing PBS. RL13 was used at 20 µg/ml, and Texas red anti-mouse IgG (Molecular Probes Inc.) and rhodamine anti-rabbit (Jackson) were diluted 1:200. Primary antibody incubations were for 1 h at room temperature; secondary antibody incubations were for 20 min.


RESULTS

We screened a cDNA expression library prepared from rat liver poly(A)mRNA with the LAP1-specific monoclonal antibody RL13 to isolate clones encoding LAP1. A single 1.9-kb positive clone was isolated from an initial screen. An oligonucleotide corresponding to the 5`-end of this clone was used to screen a second rat liver cDNA expression library, resulting in isolation of additional cDNAs. DNA sequencing indicated that one of these clones (clone CL, 2307 nucleotides long) contained a large open reading frame (ORF) with well defined start and stop sites that could encode a protein having the approximate size of LAP1C. The nucleotide sequence of clone CL and the deduced amino acid sequence of the large ORF are shown in Fig. 1. The cDNA is predicted to encode a protein of 506 amino acids with a calculated molecular weight of 56,696 and a pI of 6.42. It should be noted that an in-frame stop codon is located nine nucleotides upstream of the predicted start site, indicating that this clone encodes the amino terminus of a protein-coding region.

To validate that clone CL encoded a LAP1 isotype, we prepared polyclonal antibodies against a trpE fusion protein containing residues 120-334 of the open reading frame, as well as against a synthetic peptide encoding a second region (residues 466-479) of the ORF (see ``Experimental Procedures''). These antibodies were used to analyze various mammalian cell and recombinant protein samples by immunoblotting (Fig. 2). RL13, the monoclonal antibody that originally defined the LAP1 antigens (14) , recognized the 75-, 68-, and 55-kDa bands corresponding to LAPs1A, -1B, and -1C, respectively, in rat liver NEs (Fig. 2, RL13 panel, lane a) and in a sample of LAP1 antigens immunopurified from NEs (Fig. 2, RL13 panel, lane b). In immunopurified LAP1 antigens from cultured rat hepatoma (HTC) cells, RL13 recognized mainly LAP1C (Fig. 2, RL13 panel, lane c), the only major LAP1 isotype expressed in this cell type (14) . However, a weak LAP1A band also was seen in some experiments ( e.g. Fig. 2 , peptide panel). RL13 reacted with a number of weak bands in both rat liver and HTC cell samples, but these varied in intensity between different preparations ( e.g. compare with Fig. 7) and most likely are proteolytic products. Finally, RL13 reacted with a glutathione S-transferase fusion protein containing residues 120-334 of the ORF, validating that the ORF we have isolated contains an RL13 epitope (Fig. 2, RL13 panel, lane d).


Figure 2: Characterization of antibodies against LAP1 by immunoblotting. Blots from 10% SDS-polyacrylamide gels were incubated with the indicated antibodies (see ``Experimental Procedures'' and text). Lanes a, salt-washed rat liver NEs; lanes b, material immunoadsorbed from salt-washed rat liver NEs with RL13; lanes c, material immunoadsorbed from cultured HTC cells; lanes d, purified fusion protein containing amino acids 120-334 of LAP1C fused to glutathione S-transferase. The positions of LAPs1A-1C and the fusion protein ( FP) are indicated. comp., competition.




Figure 7: Mapping the RL13 epitope in the LAP1C sequence. Three LAP1C-derived constructs were used for in vitro transcription/translation. Products were immunoadsorbed with the monoclonal antibody RL13 and analyzed by electrophoresis on a 10% SDS-polyacrylamide gel and fluorography. Lane 1, full-length LAP1C; lane 2, NcoI-truncated LAP1C; lane 3, LAP1C from which the BbsI fragment was removed. Shown are immunoadsorbed fractions (pellets) from the three constructs and the unbound supernatant from BbsI deletion only. The asterisk marks the specific in vitro translation product specific for construct 3, which is not found in a control translation ( C). A schematic representation of the DNA constructs is presented at the bottom of the figure. The open box denotes the putative transmembrane sequence, B marks the position of the BbsI restriction sites at nt 430 and nt 962, and N shows the NcoI site at nt 1019.



Affinity-purified antibodies against residues 120-334 of the ORF of clone CL reacted with a glutathione S-transferase fusion protein containing this sequence, as expected (Fig. 2, fusion panel). The antibodies also reacted with three bands comigrating with LAPs1A-1C in isolated rat liver NEs, as well as with immunopurified LAPs1A-1C of NEs and immunopurified LAP1C of HTC cells (Fig. 2, fusion panel). Similarly, the anti-peptide antiserum against residues 466-479 reacted with three bands having the mobility of LAPs1A-1C in isolated NEs, as well as with immunoadsorbed LAPs1A-1C of rat liver NEs and immunopurified LAP1C of HTC cells (Fig. 2, peptide panel). The reaction of the anti-peptide antibodies with the LAP1 bands was specific, as antibody binding to these bands was selectively abolished by including an excess of immunizing peptide in immunoblot incubations (Fig. 2, peptide + compet. panel). Thus, polyclonal antibodies raised against two separate regions of the ORF encoded by clone CL reacted specifically with all three rat LAP1 isotypes, arguing that clone CL encodes a LAP1 species. The data also support the possibility that the three LAP1 isotypes are closely related, as previously suggested by their cross-reaction with RL13.

To further validate the identity of clone CL, we carried out immunofluorescence staining of cultured rat cells with affinity-purified antibodies against residues 120-334 of the ORF. These antibodies stained the nuclear periphery in an approximately continuous rim-like pattern (Fig. 3 c) that was identical to the labeling obtained with RL13 (Fig. 3 a). In mitotic cells where the NE was disassembled, the antibodies against residues 120-334 labeled the cytoplasm in a diffuse fashion (data not shown), similar to the labeling of mitotic cells obtained with RL13 (14) . Thus, not only do the antibodies against the ORF of clone CL react with LAP1 on immunoblots, they also specifically stain NEs in a LAP1-like fashion. Considered together, these results indicate that clone CL is a cDNA for a LAP1 isotype.


Figure 3: Immunofluorescence staining of cultured HTC cells with antibodies against LAP1. HTC cells were grown on coverslips, fixed with 4% formaldehyde, permeabilized with Triton X-100, and labeled for immunofluorescence microscopy as follows: panel a, RL13; panel b, corresponding phase image of panel a; panel c, affinity-purified antibodies against residues 120-334 of the putative LAP1C. The anti-peptide antibodies raised to residues 466-479 of putative LAP1C (Fig. 2) did not give a signal in immunofluorescence microscopy (data not shown).



The ORF encoded by clone CL was expressed by coupled in vitro transcription/translation and analyzed by SDS-gel electrophoresis (Fig. 4). The translation product was found to have a very similar mobility to pulse-labeled LAP1C that was immunoadsorbed from HTC cells (Fig. 4). Close examination showed that the pulse-labeled LAP1C from the cultured cells, which comigrated with LAP1C of rat liver NEs (data not shown), migrated slightly (1 kDa) faster than the in vitro translation product (Fig. 4, compare lanes 1 and 2). This difference was confirmed by coelectrophoresis of the two samples (Fig. 4, lane 3). Moreover, LAP1C immunoadsorbed from HTC cells that had been continuously labeled for 16 h also migrated faster than the in vitro translation product (Fig. 4, lanes 4 and 5). Hence, there is a small but reproducible difference between the gel mobility of the in vitro translation product and LAP1C from cultured hepatoma cells. It is conceivable that this is due to a posttranslational modification associated with LAP1C of HTC cells that is absent from the in vitro translation product. In any case, because the in vitro translation product migrates very close to LAP1C of HTC cells, we conclude that the ORF of clone CL very likely encodes LAP1C.


Figure 4: Analysis of a LAP1C cDNA by in vitro transcription/translation of cloned LAP1C. Lane 1, protein immunoadsorbed by RL13 from an in vitro transcription/translation reaction containing LAP1C cDNA (Fig. 1); lane 2, LAP1C immunoadsorbed from S pulse-labeled HTC cells; lane 3, mixture of the samples from lanes 1 and 2; lane 4, LAP1C immunoadsorbed from HTC cells that were labeled with S overnight; lane 5, mixture of the samples from lanes 1-4. The open arrowhead marks the RL13-reactive product from in vitro transcription/translation of cloned LAP1C; the closed arrowhead points to LAP1C immunoadsorbed from labeled cells in culture.



We have attempted to isolate cDNAs encoding LAP1A and LAP1B by nucleotide screening of several cDNA libraries but have been unable to isolate clones sufficiently large enough to encode these proteins. Nevertheless, we have isolated eight independent LAP1C-like cDNAs that are likely to contain partial length clones derived from either or both LAPs1A and -1B (see ``Experimental Procedures''). This cDNA class is identical in sequence to corresponding portions of the LAP1C clone except that it contains insertions after nucleotides 713 and 1059 of LAP1C (indicated by arrowheads in the LAP1C sequence of Fig. 1). The three clones of this group that are sufficiently long enough to include both insertion sites contained both insertions. The first insertion extends 81 base pairs and encodes 27 novel amino acids. The second insertion extends 151 base pairs, resulting in loss of residue 335 of LAP1C and addition of 51 novel amino acids (Fig. 5). Considered together, these data suggest that the different LAP1 isotypes are likely to arise from alternative splicing.

Consistent with this view, Northern blot analysis with a radiolabeled fragment of clone CL under high stringency conditions (see ``Experimental Procedures'') detected three hybridizing species in poly(A)RNA from HTC cells and rat liver at 2.25, 2.8, and 3.75 kb (Fig. 6). In rat liver, where the LAP1C protein is relatively minor compared to LAP1A, the 2.25-kb RNA species is substantially less abundant than the larger two species, while in HTC cells where the LAP1C protein is very major compared to LAPs1A and -1B, the 2.25-kb species is more abundant than the larger two species. We conclude that the 2.25-kb poly(A)RNA probably represents the LAP1C mRNA, based on the similarity of its size to clone CL and the correlation between the relative abundance of the 2.25-kb species and the LAP1C protein in different cell types. The two larger RNAs probably encode LAPs1A and -1B, which are likely to be alternative splice variants of a LAP1 transcript (see above).

Kyte/Doolittle hydrophobicity analysis revealed that most of LAP1C is hydrophilic (Fig. 1, bottom panel). It contains only a single hydrophobic stretch sufficiently long to be a membrane-spanning segment, which is between residues 311-333. This region is likely to be membrane-integrated in vivo since LAPs1A-1C all fractionate as integral membrane proteins resistant to extraction with harsh chaotrophs (14) . The LAP1C sequence contains 7 copies of the sequence (S/T)P XX, which can be a recognition site for cdc2 kinase (3) . All of these sites occur in the large hydrophilic region on the amino-terminal side of the putative transmembrane segment. We found no significant similarity between the protein sequence of LAP1C and other translated protein sequences from a data base search.

Assuming the sequence between residues 311-333 is a membrane-spanning domain, two topologies are possible for LAP1C in the inner nuclear membrane. In one case, the sequence between residues 1-311 would be on the nucleoplasmic surface of the inner nuclear membrane, and residues 334-506 would be on the lumenal surface. In a second scenario, the orientation would be reversed. We previously localized the epitope recognized by RL13 in isolated rat liver NEs and showed it to reside on the nucleoplasmic surface of the inner nuclear membrane. To map the epitope in the primary structure of LAP1C, we prepared several deletion constructs of LAP1C, expressed these by in vitro transcription/translation, and determined whether they were immunoadsorbed by RL13 (Fig. 7). Similar to the full-length LAP1C, a fragment containing residues 1-325 was immunoadsorbed by RL13, confirming the results of Fig. 2. Moreover, a construct from which residues 129-303 were deleted was unreactive with RL13 and appeared in the unbound supernatant (Fig. 7, lane 3, asterisk). Together, these data demonstrate that the RL13 epitope, which is localized on the nucleoplasmic surface of the inner nuclear membrane, occurs between residues 129-303 of LAP1C. Since the RL13 epitope was shown previously to be localized to the nucleoplasm (14) , LAP1C appears to be a type II integral membrane protein, containing a nucleoplasmic domain at residues 1-310, a transmembrane segment at residues 311-333, and a lumenal segment at residues 334-506.

We examined the expression patterns of the LAP1A-1C isotypes in several mouse cell types reflecting different states of differentiation, using affinity-purified polyclonal antibodies raised against residues 120-334 of rat LAP1C (Fig. 8). In mouse liver, three different LAP1 species were detected by immunoblotting at 82, 80, and 60 kDa (Fig. 8, LAP1 panel). A similar triplet of immunoreactive LAP1 species was expressed in mouse 3T3 cells (Fig. 8, LAP1 panel). The three mouse LAP1 bands migrate significantly slower than comparable members of the LAP1 triplet from rat liver (Fig. 8, LAP1 panel, compare rat and mouse lanes). By contrast, both lamin Band lamins A/C of mouse essentially comigrate with the homologous rat liver proteins (Fig. 8, lamin panels).


Figure 8: Analysis of LAP1 isotype expression in various cells and tissues. Shown are from salt-washed NEs from rat liver and mouse liver (labeled rat and mouse) and NE-enriched fractions isolated from mouse NIH 3T3 cells, mouse P19 teratocarcinoma cells, and mouse P19MES cells (a differentiated derivative of P19 cells). Two times the amount of P19 protein is loaded in the following lane ( 2 P19). Immunoblots were probed with affinity-purified polyclonal anti-LAP1 antibody or with guinea pig polyclonal antibodies against rat lamin Bor rat lamins A/C.



The most major LAP1 band of mouse liver was the 80-kDa species, while in 3T3 cells the overwhelming species was the fastest migrating band of the triplet (the 60-kDa band; Fig. 8 ). This is similar to rat cells, where LAP1A (the slowest migrating species) is the most prevalent band in liver, while LAP1C (the fastest migrating species) is the only highly expressed band in cultured cells (14) . Additional evidence for developmentally regulated expression of LAP1 isotypes came from comparing the LAP1 polypeptides of P19 cells, an undifferentiated teratocarcinoma cell line, with those of P19MES cells, a differentiated derivative of P19 cells (23) . As previously shown, P19 cells express lamin Bbut not lamins A/C (Ref. 28 and Fig. 8). Interestingly, P19 cells express almost exclusively the 60-kDa LAP1, while the differentiated derivative expressed a substantial amount of the 80- and 82-kDa LAP1 species as well. Considered together, these data indicate that different LAP1 isotypes, similar to different lamin isotypes, are expressed in a developmentally regulated fashion.


DISCUSSION

In this study, we have characterized rat LAP1C by cDNA cloning and topological mapping and have begun an analysis of developmental expression of LAP1 isotypes. LAP1C is predicted to consist of 506 amino acids and is hydrophilic throughout most of its length. It contains only one predicted membrane-spanning region, which is found between residues 311-333. The epitope recognized by the LAP1-specific monoclonal antibody RL13 was found to occur on the amino-terminal side of this predicted transmembrane sequence. Since the RL13 epitope is localized to the nucleoplasm (14) , LAP1C appears to be a type II integral membrane protein, with an amino-terminal hydrophilic domain of 310 residues and a carboxyl-terminal lumenal domain of 173 amino acids (Fig. 9). Seven copies of the sequence (S/T)P XX, which is a potential recognition site by cdc2 kinase (29) , are found in LAP1C, and all are localized to the nucleoplasmic domain. Thus, the mitosis-specific phosphorylation described for LAP1 (15) could involve these cdc2 sites. The function of mitotic LAP1 phosphorylation is presently unknown. However, at least by itself, mitotic phosphorylation of LAP1 does not detectably alter the binding of LAP1 to lamins in vitro (15) .

Up to now, we have been unable to isolate full-length cDNAs encoding LAPs1A and -1B. However, we have isolated cDNAs that appear to be partial length clones for one or both of these. This class of cDNAs is identical to the LAP1C sequence with the addition of two insertions. The first insertion, which is found in the nucleoplasmic domain, places 27 novel amino acids after amino acid 219 of LAP1C. The second insertion, which is found immediately on the lumenal side of the predicted transmembrane sequence, adds 51 novel amino acids after residue 334 of LAP1C and results in loss of residue 335. These data, together with our results that antibodies against two different LAP1 regions bind to all three LAP1 isotypes, and our data that three different LAP1-like mRNAs are detected on Northern blots by high stringency hybridization indicate that different LAP1 isotypes are likely to arise from alternative splicing. It should be noted that a hypothetical polypeptide containing the LAP1C sequence with these two insertions would have a mass of 65.6 kDa, which is close to the mass of LAP1B estimated from SDS-gel electrophoresis (68 kDa). However, we have not directly determined whether LAP1B is represented by the LAP1C sequence with these two insertions.

By comparing the LAP1 protein profiles in undifferentiated mouse P19 teratocarcinoma cells, the differentiated P19MES line, and mouse liver and 3T3 cells, we obtained evidence for developmentally regulated expression of different LAP1 isotypes. Assuming that the two larger MLAP1-related polypeptides of mouse correspond to LAPs1A and -1B and the smallest Mspecies represents LAP1C, our data showed that LAPs1A and -1B are expressed strongly only in the differentiated cells, similar to lamins A/C, while LAP1C is expressed in all the samples. LAPs1A and -1B appear to interact more strongly with nuclear lamins than LAP1C, based on in vitro binding of solubilized LAP1 to nuclear lamins and relative susceptibility to extraction from the nuclear lamina by nonionic detergent/salt treatment (14, 15) . Thus, expression of LAPs1A and -1B in differentiated cells could be important for promoting enhanced stability of nuclear structure.

Interestingly, a stretch of 27 residues encoded by the second sequence insertion into LAP1C that is likely to be present in LAPs1A and/or -1B (the first 27 residues of the insertion in Fig. 5 D) has a strong (>93%) probability of forming a coiled-coil -helix when analyzed by the program of Lupas et al. (32) . Coiled-coiled regions of a similar size have the capacity to induce dimerization (30) . If this sequence specifies dimerization of LAP1 polypeptides, then its presence in LAP1A and 1B but not in LAP1C could explain why LAPs1A and -1B (hypothetical dimers) bind to polymeric lamins with higher avidity than LAP1C (a hypothetical monomer).


Figure 5: Insertions found in a class of LAP1 cDNAs at two positions of LAP1C sequence. A, amino sequence of LAP1C around the first insertion site at nt 713; B, deduced amino acid sequence at the latter site containing the insertion; C, amino acid sequence of LAP1C around the second insertion site at nt 1059; D, deduced amino acid sequence at the latter site containing the insertion. In B and D, the residues derived from the original LAP1C sequence are indicated in bold typeface, and the inserted sequences are indicated in normal typeface. The sequence inserted at nt 1059 occurs at a mid-codon position, resulting in the loss of residue 335 of LAP1C ( normal typeface in C).



A number of potential functions can be envisaged for LAP1. Because of its ability to specifically bind both A- and B-type lamins, it could be involved in attaching the lamina to the inner nuclear membrane and in targeting lamins to the NE during interphase, thereby contributing to NE architecture (15) . LAP1 (similar to LAP2) becomes concentrated at the surfaces of anaphase chromosomes at early periods of nuclear membrane reassembly (15) . Therefore, it could also play a role in NE reassembly by helping to target lamins and membrane vesicles to chromosome surfaces at the end of mitosis (11) . The membrane topology of LAP1 revealed by our analysis, consisting of a single transmembrane sequence and nucleoplasmic domain of substantial size, reflects an organization that might be expected for a protein dedicated to attaching other structures to the inner nuclear membrane. Moreover, LAP1 is sufficiently abundant to carry out this function, since it is present at an estimated 10% of the molar level of individual lamins in rat liver NEs (14) . The information reported in this study is expected to provide the basis for a detailed investigation of the role of LAP1 in NE structure and function.


FOOTNOTES

*
This work was supported by a grant from the National Institutes of Health (to L. G.) and an EMBO long-term postdoctoral fellowship (to C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U19614 and U20286.

§
Present address: Pharmacia Biotech Inc., Alameda, CA 94501.

To whom correspondence should be addressed: The Scripps Research Institute, Dept. of Cell Biology, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8514; Fax: 619-554-6253.

The abbreviations used are: NE, nuclear envelope; kb, kilobase(s); ORF, open reading frame; nt, nucleotide(s); PBS, phosphate-buffered saline.

Furukawa, K., Panté, N., Aebi, U., and Gerace, L. (1995) EMBO J. 14, in press.


ACKNOWLEDGEMENTS

We are grateful to Joanne Westendorf and other members of our laboratory for helpful discussions throughout the course of this work, to Dave Byrd for providing samples of isolated nuclear envelopes, and to Christian Fritze for helpful comments on the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.