Central pore residues mediate the p97/VCP activity required for ERAD

Byron DeLaBarre; John C Christianson; Ron R Kopito; Axel T Brunger

doi:10.1016/j.molcel.2006.03.036

Central pore residues mediate the p97/VCP activity required for ERAD

Mol Cell. 2006 May 19;22(4):451-62. doi: 10.1016/j.molcel.2006.03.036.

Authors

Byron DeLaBarre¹, John C Christianson, Ron R Kopito, Axel T Brunger

Affiliation

¹ Howard Hughes Medical Institute, Stanford University, JH Clark Center E300-C, California 94305, USA.

PMID: 16713576
DOI: 10.1016/j.molcel.2006.03.036

Abstract

The AAA-ATPase p97/VCP facilitates protein dislocation during endoplasmic reticulum-associated degradation (ERAD). To understand how p97/VCP accomplishes dislocation, a series of point mutants was made to disrupt distinguishing structural features of its central pore. Mutants were evaluated in vitro for ATPase activity in the presence and absence of synaptotagmin I (SytI) and in vivo for ability to process the ERAD substrate TCRalpha. Synaptotagmin induces a 4-fold increase in the ATPase activity of wild-type p97/VCP (p97/VCP(wt)), but not in mutants that showed an ERAD impairment. Mass spectrometry of crosslinked synaptotagmin . p97/VCP revealed interactions near Trp551 and Phe552. Additionally, His317, Arg586, and Arg599 were found to be essential for substrate interaction and ERAD. Except His317, which serves as an interaction nexus, these residues all lie on prominent loops within the D2 pore. These data support a model of substrate dislocation facilitated by interactions with p97/VCP's D2 pore.

Publication types

Research Support, U.S. Gov't, P.H.S.

MeSH terms

Adenosine Triphosphatases / chemistry*
Adenosine Triphosphatases / genetics
Adenosine Triphosphatases / metabolism*
Animals
Cell Cycle Proteins / chemistry*
Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism*
Endoplasmic Reticulum / metabolism
In Vitro Techniques
Kinetics
Mice
Models, Biological
Models, Molecular
Multiprotein Complexes
Mutagenesis, Site-Directed
Nuclear Proteins / chemistry*
Nuclear Proteins / genetics
Nuclear Proteins / metabolism*
Protein Structure, Quaternary
Rats
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Synaptotagmin I / chemistry
Synaptotagmin I / genetics
Synaptotagmin I / metabolism
Transfection
Valosin Containing Protein

Substances

Cell Cycle Proteins
Multiprotein Complexes
Nuclear Proteins
Recombinant Proteins
Synaptotagmin I
Syt1 protein, rat
Adenosine Triphosphatases
p97 ATPase
Valosin Containing Protein
Vcp protein, mouse
Vcp protein, rat