cDNA Cloning and Characterization of Lamina-associated Polypeptide 1C (LAP1C), an Integral Protein of the Inner Nuclear Membrane

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. 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. The nuclear envelope (NE), 1The abbreviations used are:NEnuclear envelopekbkilobase(s)ORFopen reading framentnucleotide(s)PBSphosphate-buffered saline. the membrane boundary of the nucleus in eukaryotic cells, consists of inner and outer nuclear membranes, nuclear pore complexes, and nuclear lamina (1Gerace L. Burke B. Annu. Rev. Cell Biol. 1988; 4: 335-374Crossref PubMed Scopus (520) Google Scholar). 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 (2McKeon F. Curr. Opin. Cell Biol. 1991; 3: 82-86Crossref PubMed Scopus (64) Google Scholar, 3Nigg E. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar, 4Georgatos S.D. Meier J. Simos G. Curr. Opin. Cell Biol. 1994; 6: 347-353Crossref PubMed Scopus (75) Google Scholar). The lamina contains mainly a polymer of nuclear lamins, members of the intermediate filament protein superfamily (2McKeon F. Curr. Opin. Cell Biol. 1991; 3: 82-86Crossref PubMed Scopus (64) Google Scholar, 3Nigg E. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar). A number of less abundant polypeptides are associated with the lamina as well, including certain integral membrane proteins (see below). nuclear envelope kilobase(s) open reading frame nucleotide(s) phosphate-buffered saline. 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 B1and B2) 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 (1Gerace L. Burke B. Annu. Rev. Cell Biol. 1988; 4: 335-374Crossref PubMed Scopus (520) Google Scholar, 3Nigg E. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar). Recent studies have shown that both A- and B-type lamins bind to chromatin (5Glass J. Gerace L. J. Cell Biol. 1990; 111: 1047-1057Crossref PubMed Scopus (154) Google Scholar, 6Höger T. Krohne G. Kleinschmidt J. Exp. Cell Res. 1991; 197: 280-289Crossref PubMed Scopus (108) Google Scholar, 7Yuan J. Simos G. Blobel G. Georgatos S. J. Biol. Chem. 1991; 266: 9211-9215Abstract Full Text PDF PubMed Google Scholar) or DNA (8Shoeman R. Traub P. J. Biol. Chem. 1990; 265: 9055-9061Abstract Full Text PDF PubMed Google Scholar, 9Luderus M. de Graaf A. Mattia E. den Blaauwen J. Grande M. de Jong L. von Driel R. Cell. 1992; 70: 949-959Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 10Luderus M. der Blaauwen J. Smit O. Compton D. von Driel R. Mol. Cell. Biol. 1994; 14: 6297-6305Crossref PubMed Google Scholar). 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 (11Gerace L. Foisner R. Trends Cell Biol. 1994; 4: 127-131Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Newly synthesized lamins A, B1, and B2are isoprenylated at carboxyl-terminal Caa X motifs, and this modification is important for their targeting to the inner nuclear membrane (2McKeon F. Curr. Opin. Cell Biol. 1991; 3: 82-86Crossref PubMed Scopus (64) Google Scholar). 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 (12Marshall C. Science. 1993; 259: 1865-1866Crossref PubMed Scopus (303) Google Scholar), it is likely that integral membrane proteins also are important for the lamina-inner membrane interaction (13Hennekes H. Nigg E. J. Cell Sci. 1994; 107: 1019-1029Crossref PubMed Google Scholar). 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 (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar, 15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar) and a 54/58-kDa polypeptide found in avian and human cells (16Worman H. Yuan J. Blobel G. Georgatos S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8531-8534Crossref PubMed Scopus (299) Google Scholar, 17Worman H. Evans C. Blobel G. J. Cell Biol. 1990; 111: 1535-1542Crossref PubMed Scopus (193) Google Scholar, 18Bailer S.M. Eppenberger H.M. Griffiths G. Nigg E.A. J. Cell Biol. 1990; 110: 1489-1499Crossref PubMed Scopus (171) Google Scholar, 19Ye Q. Worman H. J Biol. Chem. 1994; 269: 11306-11311Abstract Full Text PDF PubMed Google Scholar) that is homologous to sterol C14 reductase of Saccharomyces cerevisiae (4Georgatos S.D. Meier J. Simos G. Curr. Opin. Cell Biol. 1994; 6: 347-353Crossref PubMed Scopus (75) Google Scholar, 11Gerace L. Foisner R. Trends Cell Biol. 1994; 4: 127-131Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Also, a 53-kDa protein (otefin) of the inner nuclear membrane of Drosphila cells had been described (20Padan R. Nainudel-Epszteyn S. Goitein R. Fainsod A. Gruenbaum Y. J. Biol. Chem. 1990; 265: 7808-7813Abstract Full Text PDF PubMed Google Scholar), 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 (15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar). 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. 2Furukawa, K., Panté, N., Aebi, U., and Gerace, L. (1995) EMBO J. 14, in press. Three different isotypes of LAP1 ranging in size from 55-75 kDa (LAPs1A, −1B, and −1C) have been described in mammalian cells (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar, 15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar). 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. RNA was isolated from rat livers (Sprague-Dawley males, 6-8 weeks) using the guanidinium isothiocyanate method (21Lizardi P.M. Methods Enzymol. 1983; 96: 24-38Crossref PubMed Scopus (48) Google Scholar), 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 λLAPII expression vector (by Marjorie Snead, Stratagene) using a mixture of random and oligo(dT) primers. The cDNA library contained 3.3 × 106primary clones, with a background of 5% non-recombinants. Approximately 4 × 105primary clones were screened with RL13 (α-LAP1) monoclonal antibody (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar) 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 with and used to a rat liver cDNA library from a clone for LAP1C (clone CO from clone in that it contains two insertions of sequence. The is found after nucleotide of clone and encoding amino The second is found after nucleotide of clone and in of of the sequence and of amino In to clones, CO was the method and was used for acid of this clones were found in a rat and were isolated from a rat library by using this clone as a nucleotide and/or nucleotide that of these clones contained or both of the insertions of on were to include both However, of these clones in the to cDNAs that be predicted to encode the amino-terminal of or The clone containing these two insertions had a 5′-end with of LAP1C to at of The clone a at with other cDNAs that were isolated or the in a after amino acid of LAP1C Since LAP1 isotypes the sequence predicted at residues of LAP1C as shown by with this this nucleotide is likely a cloning and a clone to this nucleotide was used for this were from clones to the were into from were in cells DNA was using the were synthesized as to sequence from both DNA were using and and poly(A)+RNA from cells and poly(A)+RNA from rat liver were to J. T. A Scholar). membrane in at for the RNA was to the membrane using The of clone CO of LAP1C was used to the was in × J. T. A Scholar), and DNA at were in × J. T. A Scholar), at and P19 cells were from and cells C. A. den de Exp. Cell Res. PubMed Scopus Google Scholar) were by and cells were in with and P19 cells were in containing and In and was on both and DNA using the to the of cells in cells to were in and with were in for at containing was to the cells for and cells were as described for cells were in with and of the of with were isolated from rat liver and were in a containing as described A. Gerace L. J. Cell Biol. PubMed Scopus Google Scholar). a from cultured cells, were into phosphate-buffered and were at × for were by and in and and of and and were in of on for were with in a were at × for in two in the and nuclear were to of containing of and A. in a the were to an of 2 and was and were out of this at × for and in were in a protein containing a of The expression was by an from clone CO into the vector D. A. J. PubMed Google Scholar). The protein contained the LAP1C region from residues two the amino acids of the CO (see and the amino acids of the The protein was expressed in E. by on an and used to an for anti-LAP1 from the the from clone CO was into second protein was on and to at to the were to the for at after of the were with and PubMed Scopus Google Scholar). to a corresponding to amino acids of LAP1C were by the to and the into Monoclonal RL13 (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar) was on protein and to at for as an of cultured cells for were by cells in and into 2 and The was on for and at × for to a rat liver were in a similar while in were with 4 of of LAP1 from these were with at were with and with proteins were in was carried out on as described PubMed Scopus Google Scholar), and was as described A. Gerace L. J. Cell Biol. PubMed Scopus Google Scholar). RL13 was used at and was with protein antibody was with residues of LAP1C was In the antibody was with lamins and lamin B were used at was used to were used to the cells on were for 4 in in and were for 4 in in were in RL13 was used at and and were antibody were for at antibody were for We screened a cDNA expression library prepared from rat liver with the monoclonal antibody RL13 to clones encoding A single positive clone was isolated from an corresponding to the 5′-end of this clone was used to a second rat liver cDNA expression in of DNA that of these clones (clone contained a large open reading frame with and that could encode a protein the size of LAP1C. The nucleotide sequence of clone and the amino acid sequence of the large are shown in The cDNA is predicted to encode a protein of 506 amino acids with a of and a of be that an is of the predicted that this clone the amino of a protein-coding that clone a LAP1 we prepared a protein containing residues of the open reading as as a encoding a second region of the (see These were used to mammalian and protein by the monoclonal antibody that the LAP1 (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar), recognized the and corresponding to −1B, and in rat liver RL13 and in a of LAP1 from RL13 In LAP1 from cultured rat cells, RL13 recognized mainly LAP1C RL13 the only LAP1 expressed in this type (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar). However, a also was in RL13 with a number of in both rat liver and these in between different with and most likely are RL13 with a protein containing residues of the that the we have isolated contains an RL13 epitope RL13 the RL13 epitope in the LAP1C sequence. Three were used for in were with the monoclonal antibody RL13 and by on a and LAP1C from which the was are from the and the from The the in for which is not found in a A of the DNA is at the of the The open the putative transmembrane B the of the at and and the site at residues of the of clone with a protein containing this as The also with with in isolated rat liver as as with of and LAP1C of cells the residues with the of in isolated as as with of rat liver and LAP1C of cells The of the with the LAP1 was as antibody binding to these was by including an of in Thus, two of the by clone specifically with rat LAP1 that clone a LAP1 The also the that the LAP1 isotypes are as suggested by their with the of clone we carried out of cultured rat cells with residues of the These the nuclear periphery in an approximately that was to the with RL13 In mitotic cells the NE was the residues the in a not similar to the of mitotic cells with RL13 (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar). Thus, not only the the of clone with LAP1 on also specifically in a these that clone is a cDNA for a LAP1 The by clone was expressed by in and by The was found to have a similar to LAP1C that was from cells that the LAP1C from the cultured cells, which with LAP1C of rat liver not the in and was by of the two LAP1C from cells that had been for also the in 4 and is a between the of the in and LAP1C from cultured is that this is to a modification associated with LAP1C of cells that is from the in In the in to LAP1C of cells, we that the of clone likely LAP1C. We have to cDNAs encoding and by nucleotide of several cDNA have been to clones large to encode these we have isolated cDNAs that are likely to clones from or both LAPs1A and (see cDNA class is in sequence to corresponding of the LAP1C clone that it contains insertions after and of LAP1C by in the LAP1C sequence of The clones of this that are to include both contained both The and amino The second in of of LAP1C and of amino acids these suggest that the different LAP1 isotypes are likely to arise from alternative splicing. with this analysis with a of clone (see in poly(A)+RNA from cells and rat liver at and kb In rat the LAP1C protein is to the RNA is substantially less abundant the two while in cells the LAP1C protein is to LAPs1A and −1B, the is abundant the two We that the poly(A)+RNA probably the LAP1C based on the of its size to clone and the between the of the and the LAP1C protein in different The two probably encode LAPs1A and −1B, which are likely to be alternative of a LAP1 (see analysis that most of LAP1C is hydrophilic contains only a single to be a membrane-spanning which is between residues 311-333. region is likely to be in as integral membrane proteins to with (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar). The LAP1C sequence contains of the sequence which be a site for E. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar). of these occur in the large hydrophilic region on the amino-terminal of the putative transmembrane segment. We found between the protein sequence of LAP1C and other protein from a the sequence between residues is a membrane-spanning two are for LAP1C in the inner nuclear In the sequence between residues be on the of the inner nuclear membrane, and residues be on the In a second the be We localized the epitope recognized by RL13 in isolated rat liver and it to on the of the inner nuclear the epitope in the of we prepared several of expressed these by in and were by RL13 to the a containing residues was by the of a from which residues were was with RL13 and in the these that the RL13 which is localized on the of the inner nuclear membrane, between residues of LAP1C. Since the RL13 epitope was shown to be localized to the nucleoplasm (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar), LAP1C to be a type II integral membrane containing a domain at residues a transmembrane segment at residues and a segment at residues We the expression of the isotypes in several mouse different of using residues of rat LAP1C In mouse different LAP1 were by at and kDa LAP1 A similar of LAP1 was expressed in mouse cells LAP1 The mouse LAP1 members of the LAP1 from rat liver LAP1 rat and mouse both lamin B1and lamins of mouse with the homologous rat liver proteins lamin The most LAP1 of mouse liver was the while in cells the was the of the is similar to rat cells, is the most in while LAP1C is the only expressed in cultured cells (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar). for developmentally regulated expression of LAP1 isotypes from the LAP1 polypeptides of P19 cells, an teratocarcinoma with of cells, a derivative of P19 cells C. A. den de Exp. Cell Res. PubMed Scopus Google Scholar). P19 cells lamin not lamins C. Burke B. Cell. Full Text PDF PubMed Scopus Google and P19 cells the while the derivative expressed a substantial of the and LAP1 as these that different LAP1 similar to different lamin are expressed in a developmentally regulated In this study, we have rat LAP1C by cDNA cloning and mapping and have an analysis of expression of LAP1 LAP1C is predicted to consist of 506 amino acids and is hydrophilic throughout most of its contains only predicted membrane-spanning which is found between residues 311-333. The epitope recognized by the monoclonal antibody RL13 was found to occur on the amino-terminal of this predicted transmembrane sequence. Since the RL13 epitope is localized to the nucleoplasm (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar), LAP1C to be a type II integral membrane with an amino-terminal hydrophilic domain of residues and a carboxyl-terminal domain of amino acids of the sequence which is a site by E. Trends Cell Biol. 1993; 3: Full Text PDF PubMed Scopus Google Scholar), are found in and are localized to the Thus, the described for LAP1 (15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar) could these The of mitotic LAP1 is However, at by mitotic of LAP1 not the binding of LAP1 to lamins in (15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar). to we have been to cDNAs encoding LAPs1A and However, we have isolated cDNAs that appear to be clones for or both of class of cDNAs is to the LAP1C sequence with the of two The which is found in the amino acids after amino acid of LAP1C. The second which is found on the of the predicted transmembrane amino acids after of LAP1C and in of These with that two different LAP1 bind to LAP1 and that different are on by that different LAP1 isotypes are likely to arise from alternative splicing. be that a polypeptide containing the LAP1C sequence with these two insertions have a mass of which is to the mass of from However, we have not is by the LAP1C sequence with these two the LAP1 protein in mouse P19 teratocarcinoma cells, the and mouse liver and cells, we for developmentally regulated expression of different LAP1 that the two polypeptides of mouse to LAPs1A and and the that LAPs1A and are expressed only in the cells, similar to lamins while LAP1C is expressed in the LAPs1A and appear to with nuclear lamins based on in binding of LAP1 to nuclear lamins and to from the nuclear lamina by (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar, 15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar). Thus, expression of LAPs1A and in cells could be important for of nuclear structure. a of residues by the second sequence into LAP1C that is likely to be present in LAPs1A and/or residues of the in has a of a by the of A. M. J. Science. 1991; PubMed Scopus Google Scholar). of a similar size have the to R. Science. PubMed Scopus Google Scholar). this sequence of LAP1 its in and not in LAP1C could LAPs1A and bind to lamins with LAP1C A number of be for of its to specifically bind both A- and B-type lamins, it could be in the lamina to the inner nuclear membrane and in targeting lamins to the NE during to NE (15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar). LAP1 to at the of chromosomes at of nuclear membrane (15Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (452) Google Scholar). it could also play a role in NE by to lamins and membrane to at the of mitosis (11Gerace L. Foisner R. Trends Cell Biol. 1994; 4: 127-131Abstract Full Text PDF PubMed Scopus (86) Google Scholar). The membrane of LAP1 by of a single transmembrane sequence and domain of substantial an organization that be for a protein to other to the inner nuclear LAP1 is abundant to out this it is present at an of the of lamins in rat liver (14Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (151) Google Scholar). The in this is to provide the for a of the role of LAP1 in NE and We are to and other members of for throughout the of this to for of isolated nuclear and to for on the