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Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF

¹ Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA
² CREST Research Project of the Japan Science and Technology Corporation, Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492 Japan

View larger version (42K): [in a new window]   Fig. 1. Mutagenesis of human emerin, targeting residues conserved with human LAP2ß. Shown are the aligned amino acid sequences of human emerin and human LAP2ß, starting with the LEM-domain of each protein (residue 1 of emerin; residue 110 of LAP2ß). Numbers on the top line refer to the LAP2ß sequence. The regions mutated in this study are indicated by lines; residues changed to alanine are indicated by A. The number below each line refers to the amino acid sequence of emerin, and names each cluster of mutations according to its most N-terminal altered residue. TMD, transmembrane domain.

View larger version (73K): [in a new window]   Fig. 2. Blot overlay assays for emerin binding to BAF or lamin A. Bacterial lysates containing wildtype (W) or mutant emerin residues 1-222 were separated on gels, blotted and probed with: (A) anti-emerin antibodies, (B) 35S-labeled BAF, or (C) 35S-labeled lamin A. Mutants are numbered according to Fig. 1.

View larger version (68K): [in a new window]   Fig. 3. Solution binding as assayed by co-immunoprecipitation. Wildtype BAF, wildtype emerin (WT, residues 1-222) and mutant emerin proteins (numbered as in Fig. 1), were synthesized and 35S-labeled in vitro using coupled transcription/translation reactions, and then immunoprecipitated using immune (lanes Em, WT, 11-214) or preimmune (pre) antiserum against emerin, or anti-BAF antisera (BAF). In vitro translation of emerin yielded a 27 kDa long form (L), and often also yielded a prominent 23 kDa short form (S) (Östlund et al., 1999), assumed to arise by translation initiation at an internal site, as well as several smaller bands.

View larger version (39K): [in a new window]   Fig. 4. Effects of disease-associated mutations S54F, 95-99 and P183H on emerin binding to BAF and lamin A. (A) Bacterial lysates containing wildtype (wt) emerin protein, disease-linked emerins (S54F, 95-99, P183H) or alanine-substitution mutants (m24 and m141) were separated on gels, blotted and probed with 35S-labeled BAF or 35S-labeled lamin A. (B) Wildtype and mutant emerin proteins were synthesized as 35S-labeled proteins in vitro, mixed with 35S-labeled BAF, and immunoprecipitated with immune (shown) or preimmune (not shown) antibodies against emerin (see Materials and Methods).

View larger version (11K): [in a new window]   Fig. 5. Functional domains of emerin defined in this study. Emerin is depicted schematically, showing the LEM-domain (LEM), transmembrane domain (TM), and position of each cluster of mutations (inverted triangles; numbered as in Fig. 1). Mutations are positioned to scale along the polypeptide sequence. Domains defined in this study are the BAF binding domain (residues 1-50, which include the LEM-domain), the lamin-binding domain (residues 70-178) and a proposed third domain of unknown function (residues 179-222). Stars indicate the positions of human mutations that cause Emery-Dreifuss muscular dystrophy. Shading at the right end of the proposed lamin-binding domain indicates less severely reduced binding of lamin A to mutant m164.

View larger version (55K): [in a new window]   Fig. 6. Localization of GFP-fused emerin mutants S54F, 95-99, P183H and P183T in living HeLa cells. HeLa cells were transiently transfected to express the indicated emerin mutant as a GFP-fusion protein. (A) GFP fluorescence during interphase. (B) GFP fluorescence in living cells 5-7 minutes after the metaphase-anaphase transition, when wildtype emerin localizes to the ‘core’ region of telophase chromosomes. Bars, 10 µm.

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