An emerin "proteome": purification of distinct emerin-containing complexes from HeLa cells suggests molecular basis for diverse roles including gene regulation, mRNA splicing, signaling, mechanosensing, and nuclear architecture.
Using recombinant bead-conjugated emerin, we affinity-purified seven proteins from HeLa cell nuclear lysates that bind emerin either directly or indirectly. These proteins were identified by mass spectrometry as nuclear alphaII-spectrin, nonmuscle myosin heavy chain alpha, Lmo7 (a predicted transcription regulator; reported separately), nuclear myosin I, beta-actin (reported separately), calponin 3, and SIKE. We now report that emerin binds nuclear myosin I (NMI, a molecular motor) directly in vitro. Furthermore, bead-conjugated emerin bound nuclear alphaII-spectrin and NMI equally well with or without ATP (which stimulates motor activity), whereas ATP decreased actin binding by 65%. Thus alphaII-spectrin and NMI interact stably with emerin. To investigate the physiological relevance of these interactions, we used antibodies against emerin to affinity-purify emerin-associated protein complexes from HeLa cells and then further purified by ion-exchange chromatography to resolve by net charge and by size exclusion chromatography yielding six distinct emerin-containing fractions (0.5-1.6 MDa). Western blotting suggested that each complex had distinct components involved in nuclear architecture (e.g., NMI, alphaII-spectrin, lamins) or gene or chromatin regulation (BAF, transcription regulators, HDACs). Additional constituents were identified by mass spectrometry. One putative gene-regulatory complex (complex 32) included core components of the nuclear corepressor (NCoR) complex, which mediates gene regulation by thyroid hormone and other nuclear receptors. When expressed in HeLa cells, FLAG-tagged NCoR subunits Gps2, HDAC3, TBLR1, and NCoR each co-immunoprecipitated emerin, validating one putative complex. These findings support the hypothesis that emerin scaffolds a variety of functionally distinct multiprotein complexes at the nuclear envelope in vivo. Notably included are nuclear myosin I-containing complexes that might sense and regulate mechanical tension at the nuclear envelope.
Holaska
James M
JM
Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Wilson
Katherine L
KL
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites / Q000502:physiology; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D046912:Multiprotein Complexes / Q000378:metabolism*; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D014176:Protein Biosynthesis; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002843:Chromatin; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C092710:emerin