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, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major "classic" heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed 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 (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive 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 cells treated at 45 degrees C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)+ RNA extracted from cells heated at 42 degrees C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro. hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer.
Delpino
A
A
Laboratory of Biophysics, Regina Elena Institute for Cancer Research, Rome, Italy.
Mileo
A M
AM
Lapenta
V
V
Piselli
P
P
Verdina
A
A
Polenzani
L
L
D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000276:immunology; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008970:Molecular Weight; D011817:Rabbits; D014407:Tumor Cells, Cultured
D018840:HSP70 Heat-Shock Proteins; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins