The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

  1. Susan R. Wente1,6
  1. 1Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA;
  2. 2Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208, USA;
  3. 3Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA
    • 4 Present addresses: Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47097, USA;

    • 5 Department of Cell Biology, Yale University, New Haven, CT 06511, USA.

    Abstract

    Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP6)-bound Gle1 to mediate remodeling of mRNA–protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP6 stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5–ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP6 regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

    Keywords

    Footnotes

    • Received February 11, 2011.
    • Accepted April 1, 2011.

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