Nuclear membrane protein MAN1: direct binding to emerin in vitro and two modes of binding to BAF

MAN1 is a vertebrate nuclear inner membrane protein that inhibits Smad signaling downstream of TGF . MAN1 has an exposed LEM-domain-containing Nterminal region (‘MAN1-N’), two transmembrane domains and an exposed Cterminal 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 transcription regulators GCL, Btf and BAF. BAF binding to this region, which has no LEM-domain, was notable. Sequence alignments identified a potential BAFbinding motif, characterized by conserved residues Ser-Arg-Val (‘SRV’), 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 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 ~40-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; 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 EmeryDreifuss 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 C. elegans (8). However in C. elegans the function of Ceemerin 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.