Heat-induced oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP.

Gp96 (GRP94) is a dimeric glycoprotein and is the endoplasmic reticulum representative of the hsp90 family of molecular chaperones. In addition to the protein substrates it chaperones, gp96 binds weakly to both peptides and ATP, and has been shown to self-assemble into discrete oligomers upon heat shock at 50 degrees C, although physiological roles for these phenomena have not been well established. Our studies indicate that gp96 homooligomerizes irreversibly in vitro at temperatures as low as 42 degrees C and could involve pre-dissociation of dimers to monomers. Oligomerization is inhibited significantly by ATP; hydrolysis is not required, since ADP, ATP-gamma-S, and NECA inhibit self-assembly equally well. Peptide ligands do not competitively inhibit gp96 self-assembly and, in fact, bind to all oligomeric species, including the dimer. Together, these findings suggest that (1) heat-enhanced chaperone activity does not reside in oligomers per se, and (2) the regions of gp96 involved in peptide binding and oligomerization are distinct.