Format

Send to

Choose Destination
See comment in PubMed Commons below
Biochemistry. 2007 Jul 31;46(30):8897-908. Epub 2007 Jul 10.

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.

Author information

1
Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.

Abstract

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.

PMID:
17620012
PMCID:
PMC2635128
DOI:
10.1021/bi602636m
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society Icon for PubMed Central
    Loading ...
    Support Center