Central pore residues mediate the p97/VCP activity required for ERAD.
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.
DeLaBarre
Byron
B
Howard Hughes Medical Institute, Stanford University, JH Clark Center E300-C, California 94305, USA.
Christianson
John C
JC
Kopito
Ron R
RR
Brunger
Axel T
AT
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; D000074405:Valosin Containing Protein; C000614179:Vcp protein, mouse; C000614180:Vcp protein, rat