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

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.

FIGURE 1

Affinity purification of emerin-associated proteins. A, Coomassie-stained gel showing proteins that bound to emerin-beads or BSA-beads. Bands a-h were identified by mass spectrometry, and are named at right. B, Immunoblots of HeLa nuclear lysate proteins (L) or proteins affinity-purified using either BSA-beads or emerin-beads in the absence of ATP. Blots were probed with antibodies to NMI, actin (all isoforms) or αII-spectrin. C, Effects of ATP treatment. Nuclear extracts were left untreated, as in (B), or treated with 10 mM ATP prior to affinity purification on BSA-beads or emerin-beads in the continuous presence of ATP. Bound proteins were resolved by SDS-PAGE and immunoblotted as in (B). D, Co-immunoprecipitation of recombinant purified emerin and NMI proteins. Recombinant NMI and emerin (residues 1-222) proteins were incubated together (L), immunoprecipitated using either immune (IM) or pre-immune (Pre) serum 2999 against emerin, and resolved by SDS-PAGE and western blotted using antibodies against NMI (α-NMI) or emerin (α-Em).

FIGURE 2

Purification of a putative emerin-NMI-actin-αII-spectrin-containing complex, and five other complexes, from HeLa cells. A, Results from a two-step purification of emerin-associated protein complexes (emerin affinity purification, ion-exchange chromatography eluted with a linear NaCl gradient). Shown is a representative Western blot of the MonoQ elution profile probed with antibodies to each indicated protein. The NaCl concentration that eluted each fraction is indicated. NL, nuclear lysate. B, The Mono-Q fraction that eluted with 0.6–0.7 M NaCl (arrow, Figure 2A) was further resolved by size exclusion (Superose 12) chromatography and fractions were Western blotted with antibodies against each indicated protein. This analysis (n=3) suggested the presence of at least two complexes: a putative 0.8–1.4 MD complex that included emerin, NMI, actin and αII-spectrin (*), and a separate complex containing emerin, NMI and actin (σ). C, Protocol used for subsequent independent purifications of emerin-containing complexes from HeLa cell nuclear lysates (n=3).

FIGURE 3

Purification of distinct emerin-containing architectural and regulatory complexes. A, Immunoblots of the proposed Architectural and Regulatory complexes show distinct protein compositions. Among candidate proteins tested, the architectural complex had detectable emerin, lamin A, lamin B, NMI and actin, whereas the regulatory complex had detectable BAF, H1, H3, p107, Lmo7, HDAC1, HDAC3 and actin. B, Analytical SDS-PAGE protein profiles of the Architectural and Regulatory complexes stained with SYPRO-Ruby to assess the number and sizes of constituent polypeptides. Proteins in each sample were identified by LCMS/MS. Selected results are described in the text and Table 1; full datasets are available upon request.

FIGURE 4

Purification of four additional emerin-containing complexes. A, Analytical SDS-PAGE analysis of complexes 24, 25, 32 and 52 stained with SYPRO-Ruby to assess the number and sizes of constituent polypeptides. Proteins in each sample were identified by LCMS/MS. B, Western blots probed for candidate components suggest complexes 24, 25, 32 and 52 have distinct protein compositions. A subset of identified components is described in the text and Table 1; full datasets are available upon request.

FIGURE 5

Emerin interacts with FLAG-tagged NCoR complex components in vivo. A,B, HeLa cells were mock transfected (−) or transfected to transiently express either FLAG-HDAC3, FLAG-TBL1, FLAG-TBLR1, FLAG-GPS or FLAG-NCoR, lysed in modified NEHN buffer and incubated with M2-agarose to immunoprecipitate FLAG-tagged proteins. Load (L, 10%), supernatant (S, 10%) and pellet (P, 50%) were immunoblotted with antibodies against the FLAG-tag (α-FLAG) or emerin (α-emerin).