The Major Nuclear Envelope Targeting Domain of LAP2 Coincides with Its Lamin Binding Region but Is Distinct from Its Chromatin Interaction Domain
1998; Elsevier BV; Volume: 273; Issue: 7 Linguagem: Inglês
10.1074/jbc.273.7.4213
ISSN1083-351X
AutoresKazuhiro Furukawa, Christian Fritze, Larry Gerace,
Tópico(s)Genomics and Chromatin Dynamics
ResumoLAP2 is an integral protein of the inner nuclear membrane which binds lamins and chromosomes and is suggested to have an important role in nuclear envelope organization. In a previous study we identified an internal 76-amino acid region of LAP2 which is required for stable targeting of the protein to the nuclear envelope. Here, we have mapped the lamin binding region of LAP2 and demonstrate that it coincides with this nuclear envelope targeting domain. In contrast, we found that the portion of LAP2 involved in binding to chromosomes resides in a separate region of the protein near its NH2 terminus. The minimal lamin binding region of LAP2 is capable of conferring stable nuclear envelope localization when attached to the transmembrane and partial lumenal domains of a protein that shows no nuclear envelope targeting activity. This directly supports the notion that a major mechanism for localization of integral membrane proteins at the inner nuclear membrane involves binding to lamins, which would constrain diffusion through the continuous nuclear envelope/endoplasmic reticulum membrane system. LAP2 is an integral protein of the inner nuclear membrane which binds lamins and chromosomes and is suggested to have an important role in nuclear envelope organization. In a previous study we identified an internal 76-amino acid region of LAP2 which is required for stable targeting of the protein to the nuclear envelope. Here, we have mapped the lamin binding region of LAP2 and demonstrate that it coincides with this nuclear envelope targeting domain. In contrast, we found that the portion of LAP2 involved in binding to chromosomes resides in a separate region of the protein near its NH2 terminus. The minimal lamin binding region of LAP2 is capable of conferring stable nuclear envelope localization when attached to the transmembrane and partial lumenal domains of a protein that shows no nuclear envelope targeting activity. This directly supports the notion that a major mechanism for localization of integral membrane proteins at the inner nuclear membrane involves binding to lamins, which would constrain diffusion through the continuous nuclear envelope/endoplasmic reticulum membrane system. The nuclear envelope (NE) 1The abbreviations used are: NE, nuclear envelope; ER, endoplasmic reticulum; LAP, lamina-associated protein; LBR, p58/lamin B receptor; HA, hemagglutinin; GST, glutathioneS-transferase.1The abbreviations used are: NE, nuclear envelope; ER, endoplasmic reticulum; LAP, lamina-associated protein; LBR, p58/lamin B receptor; HA, hemagglutinin; GST, glutathioneS-transferase. is a specialized region of the ER that forms the nuclear boundary in eukaryotes (for review, see Refs. 1Georgatos S.D. Meier J. Simos G. Curr. Opin. Cell Biol. 1994; 6: 347-353Crossref PubMed Scopus (75) Google Scholar, 2Gerace L. Burke B. Annu. Rev. Cell Biol. 1988; 4: 335-374Crossref PubMed Scopus (519) Google Scholar, 3Nigg E.A. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar, 4Rout M. Wente S. Trends Cell Biol. 1994; 4: 357-365Abstract Full Text PDF PubMed Scopus (247) Google Scholar). It consists of a double membrane that is perforated by pore complexes and which is lined by the nuclear lamina in higher eukaryotic cells. The outer nuclear membrane is continuous with the more peripheral ER and is linked to the inner nuclear membrane via a "pore membrane" adjacent to the pore complexes. Whereas the outer membrane is biochemically and functionally similar to peripheral ER, the inner membrane differs markedly from the latter because of its association with the nuclear lamina and its content of specific integral membrane proteins that are mostly absent from the peripheral ER (5Marshall I.C.B. Wilson K.L. Trends Cell Biol. 1997; 7: 69-74Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 6Gerace L. Foisner R. Trends Cell Biol. 1994; 4: 127-131Abstract Full Text PDF PubMed Scopus (86) Google Scholar). The nuclear lamina is thought to provide a structural framework for the NE and a chromatin anchoring site at the nuclear periphery (for review, see Refs. 1Georgatos S.D. Meier J. Simos G. Curr. Opin. Cell Biol. 1994; 6: 347-353Crossref PubMed Scopus (75) Google Scholar, 3Nigg E.A. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar). The lamina contains a polymer of intermediate-type filament proteins termed lamins as well as a number of more minor lamina-associated polypeptides. Four major lamin subtypes have been identified in mammalian somatic cells, lamins A, B1, B2, and C (for review, see Ref. 3Nigg E.A. Curr. Opin. Cell Biol. 1992; 4: 105-109Crossref PubMed Scopus (118) Google Scholar). In higher eukaryotes, four lamina-associated proteins of the inner nuclear membrane have been identified: lamina-associated polypeptide (LAP)1 (7Senior A. Gerace L. J. Cell Biol. 1988; 107: 2029-2036Crossref PubMed Scopus (148) Google Scholar, 8Martin L. Crimaudo C. Gerace L. J. Biol. Chem. 1995; 270: 8822-8828Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), LAP2 (9Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar), p58/lamin B receptor (LBR) (11Worman H.J. Yuan J. Blobel G. Georgatos S.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8531-8534Crossref PubMed Scopus (298) Google Scholar, 12Worman H.J. Evans C.D. Blobel G. J. Cell Biol. 1990; 111: 1535-1542Crossref PubMed Scopus (193) Google Scholar), and otefin (13Padan R. Nainudel-Epszteyn S. Goitein R. Fainsod A. Gruenbaum Y. J. Biol. Chem. 1990; 265: 7808-7813Abstract Full Text PDF PubMed Google Scholar). LAP1 (8Martin L. Crimaudo C. Gerace L. J. Biol. Chem. 1995; 270: 8822-8828Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), LAP2 (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar) and LBR (11Worman H.J. Yuan J. Blobel G. Georgatos S.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8531-8534Crossref PubMed Scopus (298) Google Scholar) are type II integral membrane proteins (14Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar). LAP1 and LAP2 each contains a single predicted transmembrane domain and a large nucleoplasmic region (8Martin L. Crimaudo C. Gerace L. J. Biol. Chem. 1995; 270: 8822-8828Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). In contrast, LBR, which is homologous to yeast sterol C14 reductase (discussed in Ref. 1Georgatos S.D. Meier J. Simos G. Curr. Opin. Cell Biol. 1994; 6: 347-353Crossref PubMed Scopus (75) Google Scholar), contains eight predicted membrane-spanning regions. Otefin appears to be more peripherally associated with the inner nuclear membrane based on chemical extraction and contains a short hydrophobic segment at its COOH terminus that is not predicted to span the inner nuclear membrane (15Ashery-Padan R. Weiss A.M. Feinstein N. Gruenbaum Y. J. Biol. Chem. 1997; 272: 2493-2499Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A fifth NE-specific protein with a putative transmembrane domain, emerin, has been identified in mammals (16Manilal S. Nguyen T.M. Sewry C.A. Morris G.E. Hum. Mol. Genet. 1996; 5: 801-808Crossref PubMed Scopus (316) Google Scholar, 17Nagano A. Koga R. Ogawa M. Kurano Y. Kawada J. Okada R. Hayashi Y.K. Tsukahara T. Arahata K. Nat. Genet. 1996; 12: 254-259Crossref PubMed Scopus (291) Google Scholar, 18Bione S. Maestrini E. Rivella S. Mancini M. Regis S. Romeo G. Toniolo D. Nat. Genet. 1994; 8: 323-327Crossref PubMed Scopus (764) Google Scholar). Emerin has two short regions of homology to LAP2 (16Manilal S. Nguyen T.M. Sewry C.A. Morris G.E. Hum. Mol. Genet. 1996; 5: 801-808Crossref PubMed Scopus (316) Google Scholar), but whether it is localized to the inner nuclear membrane has not yet been determined definitively. All three well characterized integral proteins of the inner nuclear membrane, LAP1 and LAP2 (9Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar) and LBR (11Worman H.J. Yuan J. Blobel G. Georgatos S.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8531-8534Crossref PubMed Scopus (298) Google Scholar), have been shown to bind to lamins. Through this binding interaction, they could contribute to the attachment of the nuclear lamina to the inner nuclear membrane. Lamins (19Yuan J. Simos G. Blobel G. Georgatos S.D. J. Biol. Chem. 1991; 266: 9211-9215Abstract Full Text PDF PubMed Google Scholar, 20Taniura H. Glass C. Gerace L. J. Cell Biol. 1995; 131: 33-44Crossref PubMed Scopus (234) Google Scholar, 21Hoger T.H. Krohne G. Kleinschmidt J.A. Exp. Cell Res. 1991; 197: 280-289Crossref PubMed Scopus (108) Google Scholar, 22Glass J.R. Gerace L. J. Cell Biol. 1990; 111: 1047-1057Crossref PubMed Scopus (154) Google Scholar, 23Burke B. Exp. Cell Res. 1990; 186: 169-176Crossref PubMed Scopus (67) Google Scholar) as well as LAP2 (9Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar) and LBR (24Ye Q. Worman H.J. J. Biol. Chem. 1996; 271: 14653-14656Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) bind to chromatin. At least some of these interactions are likely to promote the attachment of chromatin to the NE and higher order chromosome organization in the interphase nucleus. The ability of LAP2 to bind to lamins and chromosomes is regulated by mitotic phosphorylation (9Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar), raising the possibility that the dynamics of this protein during mitosis are closely linked to the processes of NE disassembly and reformation. LBR also is phosphorylated during mitosis (25Nikolakaki E. Meier J. Simos G. Georgatos S.D. Giannakouros T. J. Biol. Chem. 1997; 272: 6208-6213Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 26Courvalin J.C. Segil N. Blobel G. Worman H.J. J. Biol. Chem. 1992; 267: 19035-19038Abstract Full Text PDF PubMed Google Scholar), but whether this affects its ability to bind to chromatin and lamin is not known. The question of how integral proteins become targeted to the inner nuclear membrane has been raised by a number of recent studies. During mitosis when the NE is disassembled, LAP1 and LAP2 (27Yang L. Guan T. Gerace L. J. Cell Biol. 1997; 137: 1199-1210Crossref PubMed Scopus (194) Google Scholar) as well as LBR (28Ellenberg J. Siggia E.D. Moreira J.E. Smith C.L. Presley J.F. Worman H.J. Lippincott-Schwartz J. J. Cell Biol. 1997; 138: 1193-1206Crossref PubMed Scopus (627) Google Scholar) become dispersed throughout bulk ER membranes, and the NE appears to lose its identity as a distinct membrane system. Conversely these proteins become highly concentrated at the chromosome surfaces during late anaphase, when nuclear membrane reassembly around chromosomes takes place. It has been proposed that segregation of integral membrane proteins to the reforming NE at the end of mitosis is driven by binding interactions at the chromosome surfaces in combination with lateral diffusion through a continuous ER reticulum (6Gerace L. Foisner R. Trends Cell Biol. 1994; 4: 127-131Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 28Ellenberg J. Siggia E.D. Moreira J.E. Smith C.L. Presley J.F. Worman H.J. Lippincott-Schwartz J. J. Cell Biol. 1997; 138: 1193-1206Crossref PubMed Scopus (627) Google Scholar). A similar mechanism may operate during interphase to target newly synthesized integral membrane proteins to the inner nuclear membrane (6Gerace L. Foisner R. Trends Cell Biol. 1994; 4: 127-131Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 29Wiese C. Wilson K.L. Curr. Opin. Cell Biol. 1993; 5: 387-394Crossref PubMed Scopus (59) Google Scholar, 30Smith S. Blobel G. J. Cell Biol. 1993; 120: 631-637Crossref PubMed Scopus (115) Google Scholar). Alternatively, it is conceivable that integral proteins are delivered to the inner nuclear membrane by some other process that does not involve simple diffusion in the membrane bilayer. We recently identified a 76-amino acid region in the nucleoplasmic domain of LAP2 which is required for Triton-stable targeting to the NE (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). In this study we have investigated directly whether the NE targeting of LAP2 could be caused by binding interactions at the inner nuclear membrane. For this we mapped the lamin and chromatin binding regions of LAP2. We found that the region of LAP2 which is necessary and sufficient for stable NE targeting coincides with the lamin binding region but is distinct from the region involved in chromosome binding. We discuss the possibility that binding to lamins could be a major mechanism for targeting integral proteins to the inner nuclear membrane. For yeast two-hybrid studies (31Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar), members of a set of LAP2 fragments described in (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar) were inserted into theBamHI site of the GAL4-DNA binding domain vector pAS2. Full-length cDNAs for lamin B1 (NcoI-AccI fragment), lamin B2 (NcoI fragment), and vimentin (BstBI-EcoRI fragment) were subcloned into theBamHI site of the GAL4 activation domain vector pACT2 using Klenow fill-in and BglII linkers. To construct the ΔNC1-TM LAP2-lectin fusion, polymerase chain reaction primers (GCG CGA TCC TCA GCG CCC TTC AAA GTA and TGC AGA TCT GTG AGG CTC TAT AAA GGA GGC) were used to amplify the transmembrane and partial lumenal domains from a chicken hepatic lectin cDNA (32Drickamer K. Mamon J.F. J. Biol. Chem. 1982; 257: 15156-15161Abstract Full Text PDF PubMed Google Scholar, 33Drickamer K. J. Biol. Chem. 1981; 256: 5827-5839Abstract Full Text PDF PubMed Google Scholar). After gel purification, this fragment was cleaved with BamHI and BglII and subcloned into the BglII site of the HA-LAP2ΔNC1 plasmid (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). GST-LAP2 fusion constructs were generated in the pGEX-2T vector (Pharmacia Biotech Inc.). GST-LAP2ΔC1 was constructed by subcloning the BamHI ΔC1 fragment as described in Ref. 10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar into theBamHI site of pGEX-2T; it encodes amino acids 1–398 of LAP2 fused to the COOH-terminal end of GST. All remaining deletion constructs in this series derive from this parental plasmid. Deletions ΔG8, ΔG6, ΔG5, ΔG4, and ΔG3 were constructed by digestion of GST-LAP2ΔC1 with StyI, HindIII, PpuMI-NotI, NotIHindIII, and NotI, respectively, followed by fill-in with Klenow and re-ligation. Deletion ΔG1 results from digestion of GST-LAP2ΔC1 with XhoI followed by re-ligation, whereas ΔG9 comes from FseI digestion, end-polishing with mung bean nuclease, and re-ligation. A description of the two-hybrid system that was used for this work has been published elsewhere (31Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar). Briefly, Saccharomyces cerevisiae strain Y187 was transformed by the procedure of Ref. 34Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1771) Google Scholar and grown on complete minimal plates lacking Trp and Leu (−Trp −Leu medium). Induction of the lacZ reporter gene was monitored by plating on media containing 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside or direct assay of β-galactosidase activity with O-nitrophenyl-β-d-galactoside (35Reynolds A. Lundblad V. Dorris D. Keareney M. Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Protocols opinions in Molecular Biology. Greene and Wiley-Interscience, New York1993: 13.6.1-13.6.5Google Scholar). For platings, freshly grown transformants were streaked to −Trp −Leu plates containing 0.04 μg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside and 70 mm sodium phosphate, pH 7.0, and grown for 3–5 days at 30 °C. For the liquid method, overnight cultures grown in −Trp −Leu liquid were diluted, grown to A600 = 1.0, and subsequently permeabilized by SDS/chloroform and assayed for β-galactosidase activity. 5-μg epitope-tagged expression vectors were transfected into HeLa cells by SupraFect transfection (Qiagen, Chatsworth, CA). The localization of chimeric LAP2 proteins was detected by immunofluorescence with anti-HA peptide-specific monoclonal antibody (Babco, Berkeley, CA) as described previously (36Furukawa K. Hotta Y. EMBO J. 1993; 12: 97-106Crossref PubMed Scopus (204) Google Scholar). Coverslips were mounted with Slow-Fade antifade component (Molecular Probes, Eugene, OR). Slides were visualized on a Zeiss Axiophot microscope configured for epifluorescence illumination, photographed with Kodak TMAX ASA400 film, digitized with a UMAX scanner and prepared for printing on a Kodak Pictrography printer using Adobe Photoshop 3.05 software. GST-LAP2 fusion proteins were expressed in Escherichia coli BL21(pLysS). Overnight cultures were diluted 1:50 into LB medium containing 10 mmmagnesium sulfate, 1% glucose, and 100 μg/ml ampicillin at 30 °C. When the culture density reached A600 = 0.5, isopropyl-1-thio-β-d-galactopyranoside was added to 1 mm isopropyl-1-thio-β-d-galactopyranoside, and incubation was continued for 45 min. Cells were harvested by centrifugation at 4 °C, resuspended in 1/20 volume of 50 mm Tris-HCl, pH 8.0, 2 mm EDTA, 100 mm NaCl and frozen in liquid nitrogen. To prepare extracts, samples were thawed on ice, sonicated twice for 30 s, and cleared by centrifugation at 20,000 × g for 20 min. Extracts were either used directly or purified by binding to glutathione-Sepharose beads (Pharmacia) for 1 h at 4 °C, followed by washing in alternating cycles of phosphate-buffered saline and phosphate-buffered saline containing 500 mm NaCl, followed by elution in phosphate-buffered saline and 15 mmreduced glutathione (Sigma). Protein samples were displayed on 12.5% acrylamide-SDS gels and stained with Coomassie Blue to assess the relative GST-LAP2 fusion content. NRK cells were grown at 37 °C in a humidified incubator containing 5% CO2 atmosphere on coverslips in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.), 10% fetal bovine serum (Hyclone Laboratories, Logan UT), and 100 units/ml penicillin and streptomycin (Life Technologies, Inc.). To obtain populations enriched in mitotic cells, cultures were grown for 11 h in media containing 2 mmthymidine (Sigma), after which the cells were washed and incubated a further 7 h in the presence of medium lacking thymidine. Samples were removed from the incubator, washed once in a physiologic buffer (TB, Ref. 37Adam S.A. Sterne-Marr R. Gerace L. Methods Enzymol. 1992; 219: 97-110Crossref PubMed Scopus (125) Google Scholar), and then incubated for 5 min on ice in TB containing 10 μg/ml digitonin (Calbiochem). After two washes with TB and 10% bovine serum albumin to remove the digitonin, 100 μl of a glutathione affinity-purified GST-LAP2 fusion protein in TB and 10% bovine serum albumin was applied to each coverslip. After 15–20 min incubation at room temperature, samples were fixed immediately by the addition of 2 ml of 4% formaldehyde in TB for 5 min. Samples were subsequently prepared for immunofluorescence microscopy as described (27Yang L. Guan T. Gerace L. J. Cell Biol. 1997; 137: 1199-1210Crossref PubMed Scopus (194) Google Scholar), using goat anti-GST polyclonal serum (1:500, Pharmacia) as primary antibody, and fluorescein isothiocyanate-coupled mouse anti-goat antibody (1:50, Pierce) as secondary antibody. Slides were analyzed as described above for the NE targeting assay. We recently found that a region in the nucleoplasmic domain of LAP2, extending from amino acid 298 to 373, is required for targeting of LAP2 to the NE in a form that is stable to extraction with Triton X-100 (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). Because LAP2 could in principle reach the inner nuclear membrane by diffusion in the membrane bilayer from the peripheral ER (see "Discussion") and because the nucleoplasmic domain of LAP2 is able to bind to lamins and chromosomes, this suggested that the targeting of LAP2 to the inner nuclear membrane could result from binding to one of these components. To explore this possibility further, we have mapped the regions of LAP2 involved in lamin and chromosome binding to determine whether either of these comaps with the region of LAP2 involved in NE targeting. We employed the yeast two-hybrid system (31Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar) to identify the lamin binding region of LAP2. The full-length LAP2 open reading frame and a number of deletion variants were cloned into the GAL4-DNA binding domain fusion vector (Fig. 1) and were assayed in yeast in pairwise combinations with GAL4-transcription activation domain fusions containing human lamin B1 or lamin B2. In addition, certain LAP2 constructs were tested for interaction with control fusions containing the cytoplasmic intermediate filament vimentin or the yeast SNF4 protein. In this assay system, the binding of LAP2 fragments to lamin B activated a lacZ reporter gene. The activity of this gene was monitored qualitatively by direct plating on 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside indicator plates for a blue/white color reaction and then was quantitated via direct assay of soluble yeast extracts for β-galactosidase enzyme activity (35Reynolds A. Lundblad V. Dorris D. Keareney M. Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Protocols opinions in Molecular Biology. Greene and Wiley-Interscience, New York1993: 13.6.1-13.6.5Google Scholar). The results of these experiments are presented in Table I. Strains carrying the full-length LAP2 and either lamin B1 or B2 displayed blue color and high β-galactosidase levels indicative of lacZ reporter induction. Thus, LAP2 binds lamin B in the two-hybrid system in agreement with previous in vitrobiochemical studies (9Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar). No binding was observed between LAP2 and the vimentin or SNF4 controls in the colony color and β-galactosidase assays, demonstrating the specificity of this interaction. Vimentin provides an especially good control for this interaction because vimentin is a member of the intermediate filament superfamily and has some sequence similarity to lamins but is cytoplasmic and is not colocalized with LAP2 in vivo. An interaction between LAP2 and vimentin also was not observed with in vitro binding assays using purified components (9Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (447) Google Scholar).Table IInteractions between LAP2 deletion fragments and lamin BGAL4-DNA binding domain fusionGAL4-activation domain fusionLamin B1Lamin B2SNF4VimentinFL9.4 /B15.3 /B0.41 /W0.32 /WΔC110.1 /B13.0 /B0.38 /W0.22 /WΔC20.13 /W0.30 /W— /W—ΔC30.14 /W0.75 /W— /W—ΔN314.9 /B10.9 /B— /W—ΔN40.15 /W0.14 /W— /W—ΔNC12.9 /B2.1 /B— /W—ΔNC33.1 /B2.9 /B0.59 /W—ΔNC49.2 /B8.9 /B— /W— /W Open table in a new tab From analysis of constructs that involved deletions of LAP2 from the COOH terminus, we determined that the COOH-terminal border of a sequence required for lamin binding is between amino acids 296 and 398 (compare ΔC1 and ΔC2). Furthermore, from examination of constructs containing deletions of LAP2 sequences from the NH2terminus, we found that sequences upstream of amino acid 298 are dispensable for lamin binding (compare ΔN3 and ΔN4). Further analysis demonstrated that the smallest LAP2 fragment that retained significant lamin binding activity contained amino acids 298–373 (ΔNC1). LacZ activation in the ΔNC1/lamin combination was not as high as full-length LAP2/lamin (2–3 units versus 10–15 units) but still was significantly above background (0.4–0.6 unit as judged by the signal obtained with SNF4). Sequences flanking the 298–373 element resulted in an increase in the LacZ signal (compare ΔNC1, ΔN3 and ΔNC4), but it is unclear whether these sequences contribute directly to lamin binding or influence binding indirectly because of effects on protein folding. Similar to these results, sequences flanking residues 298–373 also seemed to enhance stable NE targeting (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). In summary, these experiments reveal that the major lamin binding region of LAP2 occurs between residues 298 and 373, coinciding with the region that we found previously to be required for stable targeting of LAP2 to the inner NE. Whereas the region of LAP2 between residues 298 and 373 is necessary for stable targeting of transfected deletion mutants of LAP2 to the NE in cultured cells, this segment by itself (which lacks a membrane-spanning sequence) is not sufficient for NE targeting (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). To examine the NE targeting activity of this segment of LAP2 in a more physiological structural context, we fused the LAP2 lamin binding region to a polypeptide containing the transmembrane and partial lumenal domains of chicken hepatic lectin, a type II integral membrane protein like LAP2. Chicken hepatic lectin is localized to the ER and plasma membrane and is not targeted to the NE (32Drickamer K. Mamon J.F. J. Biol. Chem. 1982; 257: 15156-15161Abstract Full Text PDF PubMed Google Scholar, 38Soullam B. Worman H.J. J. Cell Biol. 1993; 120: 1093-1100Crossref PubMed Scopus (136) Google Scholar). The lectin fragment used contains amino acids 10–91 of the lectin protein, comprising 15 amino acids of the NH2-terminal sequence, a 30-amino acid hydrophobic segment containing the transmembrane domain, and a 36-amino acid fragment of the lumenal domain. This fusion construct (ΔNC1-TM) and control full-length LAP2 (FL) were expressed as HA-tagged proteins via transfection into cultured HeLa cells, and their localization was determined by immunofluorescent staining with an anti-HA antibody (Fig. 2). The full-length LAP2 was localized in a nuclear rim staining pattern characteristic of NE proteins together with some diffuse cytoplasmic staining (−Triton panel), in agreement with our previous observations (10Furukawa K. Pante N. Aebi U. Gerace L. EMBO J. 1995; 14: 1626-1636Crossref PubMed Scopus (161) Google Scholar). Furthermore, the NE association was stable, as indicated by its resistance to extraction in a buffer containing 1% Triton X-100 and 100 mm NaCl (+Triton panel). Localization of ΔNC1-TM, the LAP2 lamin binding segment/chicken hepatic lectin chimera, revealed nuclear rim staining along with a lower level of cytoplasmic staining. The nuclear rim staining was stable to Triton extraction, indicating that the chimera was stably targeted to the NE, whereas the cytoplasmic staining was almost entirely removed by this treatment. Thus, the lamin binding region of LAP2 is sufficient to target a heterologous polypeptide containing a transmembrane and partial lumenal domain to the NE in a Triton-stable fashion. To determine whether the chromosome binding region of LAP2 can be distinguished from its major NE targeting/lamin binding domain, we mapped the region of LAP2 which interacts with chromosomes using an in situ binding assay with mitotic cells. For this, cultures of coverslip-attached normal rat kidney cells that were enriched in mitotic populations (see "Experimental Procedures") were permeabilized by treatment with digitonin and were incubated with recombinant GST fusion proteins containing various regions of LAP2 (Fig. 3). The cells were then fixed and labeled with an anti-GST antibody, and the association of the GST-LAP2 fusions with the chromosomes of mitotic cells was determined by immunofluorescence microscopy. A SDS gel displaying the recombinant GST-LAP2 fusion proteins purified on glutathione-Sepharose beads is shown in Fig. 3 B. Despite extensive efforts (see "Experimental Procedures"), we were unable to eliminate partial proteolysis of the GST-LAP2 constructs in bacteria. The amount of proteolysis ranged from <5% of total protein (ΔG1, ΔG3 in Fig.3 B) to approximately 70% (ΔG5 in Fig. 3 B). Protein amounts in each experiment were normalized such that an equal amount of each intact GST-LAP2 fusion protein was introduced into the binding assay. The results of this chromosome binding experiment are shown in Fig.4. A fusion protein containing the complete nucleoplasmic domain of LAP2 (ΔC1) strongly bound to the chromosomes of permeabilized mitotic cells, whereas no binding was detected with GST alone. The mitotic cells in these cell populations were readily detectable by phase-contrast microscopy, which clearly revealed the condensed mitotic chromosomes (e.g. arrowheads in Fig. 4, GST panel). With our experimental conditions, substantial chromosome binding was observed with as little as 15 nm GST-LAP2 fusion protein. No structures in the surrounding interphase c
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