CHARACTERIZATION OF THE MOLECULAR-CHAPERONE FUNCTION OF THE HEAT-SHOCK-COGNATE-70-INTERACTING PROTEIN

A histidine-tagged form of the recently discovered molecular chaperone , 70-kDa heat-shock cognate (Hsc70)-interacting protein (Hip), has bee n expressed in Escherichia coli and purified to near homogeneity. This protein remains soluble when expressed in E. coli. Several important properties of this chaperone have been investigated. HPLC size-exclusi on chromatography indicates that the chaperone forms a tetramer simila r to what has been reported for the native protein from rat liver cyto sol. The recombinant form of Hip did not catalyze the hydrolysis of AT P and ATP analogs, although fluorescence measurements indicated that t he chaperone recognizes anthraniloyl-dATP, anthraniloyl-ADP, and 2'-O- trinitrophenyl-ATP. The role of Hip as a molecular chaperone has been confirmed by its ability to strongly bind to the reduced, carboxymethy lated form of alpha-lactalbumin. This interaction is specific for non- native domains since native alpha-lactalbumin fails to interact with H ip. Fluorescence-anisotropy measurements indicate that reduced, carbox ymethylated lactalbumin binds Hip with a K-d of 5 mu M. Although Hip a ppears to be able to bind nucleotides and non-native proteins, it is u nable to facilitate the refolding of two denatured proteins, E. coli a lkaline phosphatase and mitochondrial malate dehydrogenase. Hip inhibi ted the refolding of alkaline phosphatase and malic dehydrogenase. Inh ibition occurred at near stoichiometric levels of Hip and could not be reversed by the addition of ATP. These results suggest that Hip may r egulate the function of the Hsp70 molecular chaperone complex in vivo and play a critical role in protein folding in the eukaryotic cytoplas m.