DIMERIZATION CHARACTERISTICS OF THE 94-KDA GLUCOSE-REGULATED PROTEIN

The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family, In this study, we expressed the ba rley (Hordeum vulgare L.) GRP94 and the alpha isoform of human HSP90 ( HSP90 alpha) in Escherichia coli and compared their dimer-forming abil ities, Native polyacrylamide gel electrophoresis revealed that GRP94 ( amino acids 69-809) and the full-length form of HSP90 alpha existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-t erminal 200 amino acids of HSP90 alpha were sufficient for the dimeriz ation, Limited proteolysis of the C-terminal half of GRP94 with thromb in revealed a 16-kDa fragment, which was derived from the C-terminus o f GRP94 through the cleavage of either the Arg710-His711 or the Arg735 -Leu736 bond. These cleavage sites were nearly, if not completely, equ ivalent to the proteolyzed region of HSP90 alpha. Their structural sim ilarity prompted us to investigate, by use of a coexpression system, t he possibility that the two proteins form a heterodimeric complex, A t wo-step affinity chromatography that specifically trapped only the com plex revealed that the C-terminal 200 amino acids of HSP90 alpha and t he C-terminal 326 amino acids of GRP94 associated with HSP90 alpha and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90 alpha. In conclusion, these resul ts indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.