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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.

Original publication

DOI

10.1016/j.molcel.2006.03.036

Type

Journal article

Journal

Molecular cell

Publication Date

05/2006

Volume

22

Pages

451 - 462

Addresses

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

Keywords

Endoplasmic Reticulum, Animals, Mice, Rats, Multiprotein Complexes, Cell Cycle Proteins, Nuclear Proteins, Recombinant Proteins, Transfection, Mutagenesis, Site-Directed, Protein Structure, Quaternary, Kinetics, Models, Biological, Models, Molecular, Synaptotagmin I, Adenosine Triphosphatases