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The AAA-ATPase VCP/p97/Cdc48 uses an internal site on inhibitor-3 (I3) to bind and remove it from protein phosphatase-1 (PP1), revealing a new mechanism for protein regulation.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • The AAA-ATPase VCP/p97/Cdc48 is crucial for protein unfolding and substrate processing.
  • Mechanisms of substrate recognition and pore insertion by VCP/p97 remain debated.

Purpose of the Study:

  • To elucidate the mechanism by which VCP/p97, with its adapter p37, recognizes and processes inhibitor-3 (I3).
  • To understand how I3 is stripped from protein phosphatase-1 (PP1) for PP1 activation.

Main Methods:

  • In vitro binding assays using VCP/p97 and its adapter p37 with inhibitor-3 (I3).
  • Site-directed mutagenesis of I3 to identify the internal recognition site (IRS).
  • In vitro Förster resonance energy transfer (FRET) assay for kinetic analysis.

Main Results:

  • VCP/p97 and p37 bind to an internal recognition site (IRS) on I3.
  • Threading of a peptide loop into the VCP/p97 channel facilitates I3 removal from PP1.
  • Mutations in the IRS abolish I3 processing both in vitro and in cellular contexts.
  • Neither the amino- nor carboxy-terminal regions of I3 are essential for its processing.

Conclusions:

  • The study identifies a novel internal recognition site (IRS) on I3 for VCP/p97 binding.
  • This mechanism explains how PP1 is released from its inhibitor for activation.
  • VCP/p97 exhibits significant plasticity in substrate threading, utilizing internal sites for recognition.