CHARACTERIZATION OF A NEW HIGH-TEMPERATURE-INDUCED 66-KDA HEAT-SHOCK PROTEIN, ANTIGENICALLY RELATED TO HEAT-SHOCK PROTEIN-72

M-14 human melanoma cells, following severe hyperthermic exposures, sy nthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major ''classic'' heat-shock proteins. This hsp 66 was not expressed f ollowing mild hyperthermic exposures sufficient to trigger the synthes is of the other heat-shock proteins. The induction of hsp 66 was obser ved also in Li human glioma cells treated at 45 degrees C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (b oth melanoma and nonmelanoma) when they were subjected to the same hyp erthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the cons titutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)(+) RNA extracted from c ells treated at 45 degrees C for 20 min was able to direct the synthes is of hsp 66 (together with hsp 72) in a message-dependent rabbit reti culocyte lysate, as well as in microinjected Xenopus oocytes. By contr ast, only the hsp 72 was expressed using Poly(A)(+) RNA extracted from cells heated at 42 degrees C for 1 h. Affinity chromatography experim ents on ATP-agarose showed that hsp 66 did not bind ATP in vitro. hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and mic rosome fraction) and in the nuclei of cells recovered from a severe he at shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly boun d to nuclear structures and could not be extracted by incubation in AT P-containing buffer. (C) 1996 Wiley-Liss, Inc.