ABSTRACT
We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.
Subject(s)
Histones/metabolism , Acetylation , Animals , Binding Sites , Blotting, Western , Cell Nucleus/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Fishes , Glutathione Transferase/metabolism , Heterochromatin/metabolism , Intracellular Membranes/metabolism , Mice , Models, Biological , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , TurkeysABSTRACT
The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.
