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Related Experiment Videos

Uncoupling Kapbeta2 substrate dissociation and ran binding.

Yuh Min Chook1, Astrid Jung, Michael K Rosen

  • 1Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.

Biochemistry
|May 30, 2002
PubMed
Summary
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Karyopherin beta2 (Kapbeta2) nuclear import relies on an acidic loop for substrate release. Disrupting this loop prevents Kapbeta2 from releasing mRNA binding proteins, revealing its crucial role in nuclear transport regulation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Structural Biology

Background:

  • Karyopherin beta2 (Kapbeta2) facilitates the nuclear import of various mRNA binding proteins.
  • Nuclear import substrate release is mediated by Kapbeta2 binding to RanGTP, causing substrate dissociation.
  • Structural data suggests an internal acidic loop in Kapbeta2 couples Ran binding to substrate release.

Purpose of the Study:

  • To investigate the molecular mechanism of Ran-GTP-mediated substrate dissociation by Kapbeta2.
  • To determine the role of Kapbeta2's internal acidic loop in coupling Ran binding and substrate release.
  • To characterize modified Kapbeta2 proteins with altered acidic loops regarding their binding affinities and functional activity.

Main Methods:

  • Proteolytic cleavage and site-directed mutagenesis to create modified Kapbeta2 proteins (cl-Kapbeta2 and TL-Kapbeta2).

Related Experiment Videos

  • NMR spectroscopy to map the Kapbeta2 binding site for the mRNA binding protein A1.
  • Quantitative affinity measurements for Kapbeta2 interactions with RanGTP and substrate.
  • In vitro nuclear import assays using digitonin-permeabilized HeLa cells to assess substrate translocation.

    • Main Results:

    • Both cl-Kapbeta2 and TL-Kapbeta2 mutants are defective in Ran-mediated substrate dissociation.
    • Modified Kapbeta2 proteins exhibit comparable binding affinities for RanGTP and substrate as native Kapbeta2.
    • The TL-Kapbeta2 mutant can bind both RanGTP and substrate simultaneously, forming a ternary complex, indicating spatially distinct binding sites.
    • Nuclear import assays show a significant loss of Ran-mediated nuclear uptake of fluorescent substrate with the TL-Kapbeta2 mutant.

    Conclusions:

    • The acidic loop of Kapbeta2 is essential for coupling RanGTP binding to substrate dissociation.
    • Perturbation of the acidic loop disrupts the allosteric communication between the Ran-binding and substrate-binding sites.
    • These findings support a model where the acidic loop acts as a physical linker transmitting conformational changes from RanGTP binding to promote substrate release.