Direct Binding of Nuclear Membrane Protein MAN1 to Emerin in Vitro and Two Modes of Binding to Barrier-to-Autointegration Factor*

MAN1 is a vertebrate nuclear inner membrane protein that inhibits Smad signaling downstream of transforming growth factor β. MAN1 has an exposed LEM domain-containing N-terminal region (“MAN1-N”), two transmembrane domains, and an exposed C-terminal domain (“MAN1-C”). Many regions of human MAN1 are homologous to emerin, a LEM domain nuclear protein, loss of which causes Emery-Dreifuss muscular dystrophy (EDMD). To test the hypothesis that MAN1 function might overlap with emerin, we tested different polypeptide fragments of MAN1 for binding to selected partners of emerin. Our findings support this hypothesis. Blot overlay assays and co-immunoprecipitation studies showed that MAN1-C binds the transcription regulators GCL, Btf, and barrier-to-autointegration factor (BAF). BAF binding to this region, which has no LEM domain, was notable. Sequence alignments identified a potential BAF-binding motif, characterized by the conserved residues Ser-Arg-Val, in MAN1-C and two other BAF-binding proteins. The other region, MAN1-N, bound directly to BAF, lamin A, and lamin B1, supporting functional overlap with emerin. Unexpectedly, three independent assays showed that MAN1-N also bound directly to emerin. Proposed MAN1-emerin complexes are discussed in the context of EDMD disease mechanisms and potential in vivo functions.

The "LEM domain" is a conserved ϳ40-residue folded motif that defines a family of nuclear proteins (1,2). Vertebrate family members include LAP2␤, emerin, and MAN1 at the nuclear inner membrane and LAP2␣ in the nuclear interior (3) plus several uncharacterized human proteins provisionally named LEM2, LEM3, LEM4, and LEM5 (2). The only known function of the LEM domain is to bind directly to a conserved chromatin protein, barrier-to-autointegration factor (BAF) 1 (4). LEM domain proteins and BAF are conserved among multicellular animal eukaryotes but absent from yeast and plants. All characterized LEM domain proteins bind to nuclear intermediate filament proteins named lamins (5). In humans, loss of emerin or dominant mutations in A-type lamins cause Emery-Dreifuss muscular dystrophy (EDMD) (6,7). EDMD is characterized by early contractures of the Achilles, elbow, and neck tendons, slow degeneration of skeletal muscles, and conduction system defects in the heart that can be fatal. Emerin itself is not essential for viability in either humans or Caenorhabditis elegans (8). However, in C. elegans the function of Ce-emerin overlaps with "Ce-MAN1" (9), the only other membrane-anchored LEM domain protein in C. elegans, which is homologous to human MAN1 but orthologous to human LEM2 (see below). Thus, an understanding of the EDMD disease mechanism will require knowing which functions of human emerin are unique and which overlap with MAN1 or other LEM domain proteins. MAN1 was first identified by autoantibodies from a patient with collagen vascular disease (1). MAN1 localizes to the nuclear inner membrane and has two transmembrane domains, exposing both the N-and C-terminal domains (MAN1-N and MAN1-C, respectively) to the nucleoplasm (10). In addition to MAN1 (911 residues), the human genome also encodes an uncharacterized homologous protein named LEM2 (503 residues, Fig. 1). The LEM domain (Fig. 1, hatched) and three other regions (Fig. 1, shaded as A, C, and D) are conserved between human MAN1 and human LEM2. Domain D (105 residues) in the membrane-proximal half of MAN1-C is 54% identical between MAN1 and human LEM2. Domain A (170 residues) and luminal domain C (131 residues) are conserved to lesser extents. Because human LEM2 and the C. elegans lem-2 gene product (Ce-MAN1 protein) are similar in length (503 and 500 residues, respectively) and both lack domains B and E (apparently unique to human MAN1), we concluded that Ce-MAN1 is orthologous to human LEM2, not human MAN1 as originally thought. Thus, the functional overlap between Ce-emerin and Ce-MAN1 in C. elegans (shared binding to Ce-lamin and Ce-BAF and synthetic lethality in ϳ100-cell embryos) (9) predicts similar overlap between human emerin, LEM2, and possibly MAN1. However, the possibility of functional overlap between human emerin and human MAN1 had not been tested.
In Xenopus embryos, the proposed MAN1 alleles named XMAN1 and SANE (Smad1 antagonistic effector) regulate dorsal-ventral axis determination (3,11,12). The C-terminal domain of XMAN1 antagonizes signaling by bone morphogenetic proteins by binding directly to Smad1, Smad5, or Smad8, which are downstream effectors of signal transduction pathways triggered when BMP-4 binds its receptor at the plasma membrane (3). These results suggest XMAN1 inhibits Smad-dependent changes in gene expression (11). Similar roles are proposed for human MAN1, which binds Smads and regulates signaling downstream of the bone morphogenetic proteins and other members of the transforming growth factor ␤ superfamily of proteins (13,14). Heterozygous loss-of-function mutations in MAN1 cause syndromes characterized by increased bone density in humans (13).
We tested five known partners of emerin for binding to the N-or C-terminal domains of MAN1. Our results show that human emerin and MAN1 have extensive functional overlap and unexpectedly revealed direct binding between emerin and MAN1, implicating MAN1 as functionally relevant to the EDMD disease mechanism.

MATERIALS AND METHODS
Expression and Purification of MAN1-C and Antibody Production-A cDNA encoding the MAN1-C polypeptide (residues 650 -910) was PCRamplified from a HeLa cell cDNA library using forward (5Ј-GCC TCG AGC GTT ACA TGA AAT ATC GAT GG-3Ј) and reverse (5Ј-GCC TCG AGT CAG GAA CTT CCT TGA GAA TT-3Ј) primers. The resulting 805-bp fragment was digested with XhoI and cloned into the XhoI sites in the pET15b vector (Novagen). After sequence verification, the construct was transformed into E. coli strain BLR(DE3)pLysSpRARE. Transformed cells were grown to an A 600 of 0.6, and MAN1-C expression was induced by 1 mM isopropyl 1-thio-␤-D-galactopyranoside for 3 h. Cells were pelleted at 5,500 ϫ g and resuspended in sonication buffer (SB; 50 mM sodium phosphate, pH 8.0, 300 mM NaCl) plus phenylmethylsulfonyl fluoride (0.5 mM) and pepstatin (1 g/ml). Cells were disrupted by pulse sonication on ice. The lysate was clarified by centrifugation at 20,000 ϫ g for 20 min, and the supernatant was loaded onto nickel-nitrilotriacetic acid-agarose beads equilibrated with SB. The column was washed with 10 ml of SB containing 10 mM imidazole, and bound proteins were eluted in SB containing 200 mM imidazole. Purified MAN1-C polypeptide was used as an antigen to generate rat serum 4279 against MAN1-C (Covance, Inc., Denver, PA).
Microtiter Assays-Purified recombinant His-tagged prelamin A and lamin B1, BAF, or MAN1-C (2 g/well) or BSA (as negative control) were adsorbed to microtiter wells overnight at 4°C as described (15) in binding buffer (BB; 20 mM Hepes, pH 7.4, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA). BB containing 3% BSA was then added to block nonspecific binding sites. Wells were not allowed to dry. A 35 S-labeled probe (GCL, Btf, BAF, or MAN1 polypeptides) synthesized as described above was added to the wells and incubated overnight at 4°C. Each experiment (n ϭ 3-4) was done in triplicate. After three washes with BB, bound proteins were eluted with 5% SDS and quantified by scintillation.
Affinity Bead Assays-Purified recombinant human emerin (residues 1-222) and MAN1-C (residues 650 -910) were covalently linked to Affi-Gel-10 beads and Affi-Gel-15 beads (Bio-Rad), respectively, per manufacturer's instructions. Beads were washed three times in immunoprecipitation (IP) buffer (150 mM NaCl, 50 mM Tris, pH 8, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml leupeptin). Emerin beads were incubated with HeLa cell lysates (1 g of protein) overnight at 4°C and washed three times with IP buffer. Bound proteins were eluted by boiling samples in 2ϫ SDS sample buffer, resolved by SDS-PAGE, and immunoblotted using rat anti-MAN1 polyclonal serum 4279 at 1:1000 dilution. MAN1-C beads were incubated with 35 S-labeled GCL (500,000 counts) overnight at 4°C. Beads were washed three times with IP buffer, and bound proteins were eluted by boiling samples in 2ϫ SDS sample buffer, resolved by SDS-PAGE, and exposed to x-ray film.

RESULTS
The aligned amino acid sequences of human MAN1 and emerin revealed the conserved LEM domain as expected ( Fig.  2A, boxed) plus a region (residues 185-385) homologous to the lamin-binding region in emerin ( Fig. 2A, underlined) (16). Three regions appeared unique to MAN1: residues 50 -184, residues 386 -548, and the C-terminal domain (Fig. 2, A and B). For further study, we focused on the full N terminus (designated MAN1-N), the N terminus minus the LEM domain (N⌬LEM), the LEM domain alone (LEM), the potential laminbinding fragment (MAN1-mid), and the entire C-terminal domain (MAN1-C) as diagrammed in Fig. 2B. Each polypeptide was either expressed in bacteria (MAN1-C) or synthesized and 35 S-labeled in eukaryotic transcription-translation extracts (see "Materials and Methods") for the binding studies below.
MAN1 Binds the Globular Tails of Lamins A and B1-Lamins anchor and retain many proteins at the nuclear inner membrane (17). The MAN1-N polypeptide is both necessary and sufficient to localize MAN1 at the nuclear envelope in mammalian cells (10) and includes the region similar to the lamin-binding domain of emerin, suggesting MAN1-N might bind lamin(s). We therefore tested the MAN1-N, MAN1-mid, and LEM polypeptides for binding to lamins, specifically the C-terminal tail domains of lamin B1 and prelamin A. Purified recombinant lamin tails (or BSA as a negative control) were immobilized in microtiter wells and probed with 25 pmol of 35 S-labeled MAN1-N, 35 S-labeled MAN1-mid, or 35 S-labeled LEM domain (as a negative control). The LEM domain did not bind significantly to lamins as expected (Fig. 3A, LEM). MAN1-N bound both the prelamin A and lamin B1 tails at levels significantly above the BSA background ( Fig. 3A, N). We concluded that human MAN1 can bind directly to both A-and B-type lamins in vitro via the lamin tail domain. The MAN1mid fragment was insufficient to bind lamins above the BSA background ( Fig. 3A), suggesting that the flanking regions in MAN1-N are required for this interaction. To test lamin binding by an independent method, we resolved the lamin tails by SDS-PAGE; they were either stained with Coomassie Blue or transferred to membranes and probed with 35 S-labeled MAN1-N (Fig. 3B). As controls, each gel included lysates from bacteria expressing either wild-type (WT) emerin (residues 1-222; Fig. 3B) or a disease-causing emerin mutant (⌬95) that lacks residues 95-99 and fails to bind lamins (16). 35 S-Labeled MAN1-N did not bind the emerin mutant ⌬95 but recognized both lamin tails, particularly the B1 tail (Fig. 3B), independently confirming that MAN1 binds directly to both A-and B-type lamins. Surprisingly, MAN1-N also specifically recognized wild-type emerin (Fig. 3B); this interaction was investigated further below (see Fig. 6).
C-terminal Domain of MAN1 Binds Transcription Regulators GCL and Btf-Emerin interacts with several transcription regulators including GCL and Btf (18,19). We therefore tested MAN1 for direct binding to GCL in pulldown assays, focusing on the C-terminal region of MAN1, which binds Smad proteins (see the Introduction). 35 S-Labeled GCL was incubated with recombinant MAN1-C conjugated to Affi-Gel beads or BSAconjugated beads as the negative control. The MAN1-C beads bound GCL significantly above the BSA background (Fig. 4A). The equilibrium binding affinity between MAN1-C and GCL was measured in microtiter assays in which immobilized recombinant MAN1-C (3.18 pmol) was probed with increasing concentrations of 35 S-labeled GCL (Fig. 4B, see "Materials and Methods"). Wells containing immobilized BSA served as nega- tive controls and were subtracted to yield the curve shown in Fig. 4B. The affinity of MAN1-C for recombinant GCL in this assay averaged 364 nM (range 211-492 nM, n ϭ 4; 492 nM curve shown in Fig. 4B), about 12-fold weaker than the affinity of emerin for GCL (30 nM, range 20 -60 nM) (19).
To test possible binding to Btf, recombinant purified MAN1-C (1 g) or lysates from bacteria expressing either wildtype emerin (positive control) or the LEM domain of MAN1 (negative control) were resolved by SDS-PAGE and either stained with Coomassie Blue (Fig. 4C, lower panel) or transferred to membranes and probed with 35 S-labeled Btf (Fig. 4C,  upper panel). 35 S-Labeled Btf recognized MAN1-C and wildtype emerin but not the LEM domain (Fig. 4C, upper panel). Microtiter assays showed MAN1-C bound Btf with an equilibrium affinity of 211 nM (range 124 -312 nM, n ϭ 3; Fig. 4D). Interestingly, MAN1-C (this work) and emerin (18) bind Btf with comparably high affinities of 211 and 100 nM, respectively. Collectively, these findings showed that two emerin-binding transcription regulators (GCL and Btf) can also bind MAN1, strongly supporting the "overlapping functions" model. Because the C-terminal region of MAN1 binds directly to three different gene regulatory proteins (GCL and Btf shown here and Smads as shown by others (3)), we concluded that the C-terminal domain of MAN1 has a general role in gene regulation. Furthermore Btf binding to MAN1 might be specifically relevant to EDMD disease (see "Discussion").
The N-and C-terminal Regions of MAN1 Each Bind BAF-We used microtiter assays to test MAN1 for binding to BAF. Recombinant BAF (10 pmol) or BSA (as a negative control) were immobilized in microtiter wells and incubated with equimolar amounts of each 35 S-labeled MAN1 polypeptide. The "mid" fragment did not bind significantly above the BSA background as expected (Fig. 5A). Also as expected, the LEM domain and MAN1-N each bound BAF at levels ϳ4-fold above the background binding to BSA-containing wells (Fig. 5A, LEM   and N, respectively). The LEM domain was largely responsible for the BAF binding activity of MAN1-N because the N⌬LEM signal was reduced to less than 2-fold above background (Fig.  5A, N⌬LEM). Interestingly, MAN1-C bound BAF at levels ϳ4.8-fold above background (Fig. 5A, C), comparable with the LEM domain. Thus, BAF binds independently to two distinct functional domains of MAN1: the LEM domain and the "gene regulatory" C-terminal domain. We therefore considered the possibility that BAF might have two distinct interaction surfaces for MAN1.
To map BAF residues important for binding to MAN1-C, we tested MAN1-C binding to 19 BAF missense mutants previously characterized for their effects on binding to emerin (the LEM domain), DNA (20,21), and histones. 2 MAN1-C or BSA was immobilized on microtiter wells and probed with each 35 S-labeled BAF mutant or wild-type BAF. Ten BAF missense mutants (K6A, K6E, R8E, D9A, G25E, I26A, L46E, G47E, V51E, and W62A) had significantly reduced binding to MAN1-C (Fig. 5B). The remaining mutants bound MAN1-C at least 50% as well as wild-type BAF. The positions of wild-type BAF residues implicated in MAN1-C binding were mapped on the atomic structure of the BAF dimer (22) (Fig. 5C). Viewed from the "front" (Fig. 5C, top row), BAF residues critical for binding MAN1-C clustered in two regions: the top "shoulders" (green wild-type residues Lys-6, Arg-8, Asp-9, Gly-25, Ile-26) and the bottom surface of the dimer (green wild-type residues Val-51 and Trp-62). Residues Leu-46 and Gly-47, buried at the dimer interface, are not surface-exposed in the crystal structure and are therefore not shown. Residue Lys-53, which is critical for BAF binding to the LEM domain of emerin (20), was not essential for BAF binding to MAN1-C; mutant K53A had wild-type binding activity, and K53E reduced binding by ϳ50% (shown in blue, Fig. 5C). Residues Lys-6, Arg-8, and Asp-9 were essential to bind MAN1-C but are not essential to bind the LEM domain in emerin (20). Mutation P14A slightly but consistently enhanced binding to MAN1-C, but we do not know if this is significant (Fig. 5C, red residue). Thus, BAF may "see" the LEM and C-terminal domains of MAN1 in distinct ways (see "Discussion").
MAN1-C has no LEM domain. We therefore hypothesized that BAF might recognize a novel motif conserved in other BAF-binding proteins that lack LEM domains, such as Crx (and related homeodomain transcription activators (23, 24)), and certain histones including linker histone H1.1. 2 Pairwise alignments of MAN1-C with Crx and histone H1.1 revealed one region comprising residues 728 -735 (SRVrtetR) in MAN1 (Fig.  5D). This SRV motif was conserved in Crx and H1.1 as the consensus sequence S(R/K)Vx(t/v)x(t/f)(R/K). We propose that this motif might mediate binding to BAF (see "Discussion").
MAN1 Binds Directly to Emerin-To explore possible direct binding between emerin and MAN1, we first used purified recombinant wild-type emerin (residues 1-222) covalently linked to Affi-Gel-10 beads or BSA-linked beads to affinitypurify proteins from HeLa cell lysates. Beads were incubated overnight with HeLa cell lysates and then washed and pelleted. Bound proteins were resolved by SDS-PAGE, transferred to membranes, and probed with immune serum 4279 against human MAN1 (Fig. 6A; see "Materials and Methods"). Endogenous MAN1 has a predicted mass of 100 kDa but migrates on gels at ϳ80 kDa (1,10). Serum 4279 antibodies detected endogenous MAN1 bound specifically to emerin beads but not BSA beads (Fig. 6A), consistent with the direct binding of MAN1 to emerin seen in blot overlays (Fig. 3B). Direct binding was further tested using emerin or BSA beads incubated with 35 S-labeled MAN1-N or wild-type 35 S-labeled BAF as the positive control. Both MAN1-N and BAF bound to emerin beads but not BSA beads (Fig. 6B). Based on these three independent assays (blot overlay in Fig. 3B, affinity purification from HeLa cell lysates in Fig. 6A, and radiolabeled proteins in Fig. 6B) we concluded that MAN1-N binds directly to emerin in vitro.
The domains in emerin required to bind other partners (BAF, lamin A, transcription repressors, and actin) were previously mapped using a collection of Ala substitution mutations or selected EDMD disease-causing mutations in emerin (16,18,19,25,26). We used these mutants in blot overlay experiments to map the MAN1-binding region(s) in emerin. Lysates from bacteria expressing wild-type or mutant emerin (residues 1-222) were resolved in duplicate SDS-PAGE gels and either stained with Coomassie Blue to control for protein loading or transferred to membranes and probed with 35 S-labeled MAN1-N (Fig. 6C). MAN1-N recognized emerin mutants m11, 3 m24, m30, m34, E40K, and the disease-causing missense mutants S54A and P183H at levels similar to wild-type emerin. Binding was reduced or undetectable to two other diseasecausing mutants ⌬95 and Q133H and the Ala substitution mutants m45A, m45E, m61, m70, m76, m112, m122, m145, m151, m161, m164, m175, m179, m192, m196, m198, m206, m207, m214, and m217 (Fig. 6C). These results are mapped schematically in Fig. 6D relative to the previously tested binding partners BAF, lamin A, GCL, Btf, and actin. The proposed MAN1-binding region encompassed most of emerin outside the LEM domain, similar to the actin-binding region in emerin. DISCUSSION The ability of BAF to bind LEM domains is well established, and we show here that MAN1 is no exception. We further report that BAF also binds the C-terminal domain of MAN1. C-terminal binding involves a number of surface residues in BAF not involved in binding the LEM domain (20). The wildtype BAF residues Lys-6, Arg-8, Asp-9, Gly-25, and Ile-26 (which cluster on the top and "shoulders" of the BAF dimer) and Val-51 and Trp-62 (on the bottom surface) appear critical to bind MAN1-C. In contrast, the BAF residues Lys-6 and Arg-8 are not essential to bind the LEM domain in emerin (20). Similarly, Lys-53, which is critical to bind LEM domains, is not essential for MAN1-C. However, the D9A mutation reduces binding to both. Therefore BAF may see the LEM domain and C-terminal region in MAN1 in overlapping but distinct ways. BAF was previously shown in blot overlay assays to bind the C-terminal domain of the C. elegans lem-2 gene product (Ce-MAN1) (9). Ce-MAN1 and its human ortholog, LEM2, both lack the MAN1-unique "domain E" (Fig. 1), suggesting BAF might recognize domain D, common to all three proteins.
Importantly, we identified a motif, S(R/K)Vx(t/v)x(t/f)(R/K) that is conserved in MAN1-C and two unrelated proteins, Crx and histone H1.1, which also lack LEM domains but bind BAF. Crx is a homeodomain transcription activator; its SRV motif (residues 81-88) is located in the BAF-and DNAbinding region of Crx (residues 34 -107) (24). The E80A mutation in Crx, which alters a single residue flanking the SRV motif, disrupts Crx binding to BAF in vitro but does not disrupt Crx binding to DNA or Crx-activated gene expression (24). The SRV motif in histone H1.1 (residues 124 -131) is located within the minimal region required to bind BAF (residues 108 -215). 2 It is noteworthy that the SRV motif is also present in domain D, shared by Ce-MAN1 (data not shown) and human MAN1 (see Figs. 1 and 2A), which might account for the binding of Ce-MAN1 to BAF. We are currently mutating the SRV motif in MAN1-C to test its hypothetical role in binding to BAF. Regardless of motif, it is interesting to consider why BAF binds two different regions of MAN1. We  Fig. 2B), washed, counted, and graphed as the ratio (-fold binding) of each MAN1 probe to BAF compared with BSA control. B, microtiter assay results for MAN1-C binding to WT BAF or each indicated BAF missense mutant. Purified recombinant MAN1-C protein or BSA (negative control) was immobilized in microtiter wells, incubated with 35 S-labeled BAF (wild-type or mutant), washed, counted, and graphed relative to the binding of wild-type BAF to MAN1-C. Bars in A and B indicate standard deviations. C, atomic structure of BAF (22) showing BAF residues implicated in binding MAN1-C. Red, potentially enhanced binding; green, little/no binding; dark blue, ϳ50% reduced binding. D, partial amino acid sequences of BAF-binding regions of MAN1-C, Crx, and histone H1.1 reveal a conserved S(R/K)Vx(t/v)x(t/f)(R/K) (SRV) motif, proposed to mediate binding to BAF. ners, including BAF, A-and B-type lamins, and the transcription regulators GCL and Btf. It will be interesting in the future to determine whether emerin, like MAN1, also binds Smad proteins. Understanding the limits of functional overlap, specifically partners that bind emerin but not MAN1, will require further analysis of additional emerging partners for emerin such as splicing factor YT521-B, actin, and nesprins (25)(26)(27). MAN1 did not have the same affinity as emerin, for all transcription factors tested. Specifically, MAN1-C binds GCL more weakly than emerin (affinities of 364 and 30 nM, respectively). This difference might be meaningful in vivo, given the presence of competing partners with higher affinity for either emerin or MAN1 (see below). However, MAN1 and emerin have similar strong affinities for Btf (211 and 100 nM, respectively), a transcription regulator. Binding between Btf and emerin appears to be relevant to EDMD disease because the EDMD-causing missense mutation in emerin, S54F, selectively disrupts emerin binding to Btf but not GCL or other tested partners (18). We are intrigued by the possibility that Btf might have even stronger affinity for joint emerin-MAN1 complexes.
Our results predict that emerin and MAN1 can form functional complexes in vivo. Consistent with this idea, MAN1 and emerin are both expressed (detectable at the level of mRNA or protein) in essentially all tissues tested (1, 28). More specifically, mRNAs for both emerin and MAN1 are present in skeletal muscle and heart (1,28), the tissues primarily affected in EDMD. Direct support for the existence of emerin-MAN1 complexes comes from a recent analysis of native emerin-containing complexes purified from HeLa cells. 4 One 500-kDa complex includes emerin, MAN1, lamin A, and BAF, suggesting that supramolecular complexes containing both emerin and MAN1 exist in vivo. MAN1 is also present in at least two other emerin-containing complexes but is undetectable in proposed "architectural" complexes that contain emerin, actin, and spectrin 4 (26).
The proposed co-binding of MAN1 and emerin has interesting implications for their in vivo functions. Their interac-4 J. M. Holaska and K. L. Wilson, unpublished observations. FIG. 6. MAN1 binds emerin directly. A, purified recombinant human emerin (Em, residues 1-222) or BSA was coupled to Affi-Gel beads and incubated with protein lysates from HeLa cells. Pellets were resolved by SDS-PAGE, transferred to membranes, and immunoblotted using the anti-MAN1 serum 4279. B, emerin beads or BSA beads were incubated with 35 S-labeled MAN1-N or 35 S-labeled BAF and washed; bound proteins were resolved by SDS-PAGE, dried, and exposed to x-ray film. The autoradiogram is shown. Asterisk indicates presumed breakdown product of 35 S-labeled MAN1-N. C, crude lysates from bacteria expressing either WT or mutant emerin proteins (numbered as described in Ref. tion may positively or negatively regulate binding to shared or unique partners in vivo and thus regulate multiple downstream pathways. For example, transcription regulators (GCL, Btf) might bind more tightly to emerin-MAN1 complexes than to either protein alone. Alternatively, transcription regulators such as GCL, which binds preferentially to emerin, might disassemble the complex and liberate MAN1 for other functions. A related possibility is that emerin might positively or negatively regulate MAN1 binding to Smads. In this case, loss of emerin might disrupt MAN1-dependent Smad signaling in EDMD patients. Notably, our work showed that the EDMD disease-causing mutations ⌬95 and Q133H in emerin each abolished binding to MAN1. Although the ⌬95 mutation disrupts binding to all of the partners of emerin (6), the Q133H mutation is noteworthy because it selectively abolishes binding to MAN1 (our work) and actin (26) but no other tested partners in vitro. Future work must aim to understand (a) how interactions between MAN1 and emerin affect their in vivo functions and (b) whether MAN1-emerin interactions regulate the EDMD phenotype.