Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly

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.