Heat shock-induced phosphorylation of GroEL alters its binding and dissociation from unfolded proteins.

During heat shock of Escherichia coli, the expression of the major molecular chaperone, GroEL, increases; in addition, a small fraction of the GroEL becomes phosphorylated (Sherman, M. Yu., and Goldberg, A. L. (1992) Nature 367, 166-1692). This heat shock-induced phosphorylation was found to enhance 50-100-fold the capacity of GroEL to bind to several denatured proteins, including casein and fetuin. The phosphorylated species in the cell extract bound quantitatively to affinity columns containing these ligands, and treatment of the extract with alkaline phosphatase markedly reduced this binding. Like heat shock (42 degrees C), overproduction of GroEL (5-10-fold) from the multicopy plasmid at low temperature (25 degrees C) increased the phosphorylated fraction, which bound strongly to denatured fetuin. Heat shock of these cells further enhanced phosphorylation, and about 15% of the induced level of GroEL could bind tightly to the fetuin column. The predominant form of the GroEL that bound to the denatured protein columns appeared to contain at least one phosphate on each of its subunits, although multiple phosphorylated subunits were also observed. With fetuin and casein as affinity ligands, only the phosphorylated species bound, and this material dissociated quantitatively upon addition of ATP-Mg2+. With CRAG and histone as the ligands, some unphosphorylated GroEL also bound, but this species (unlike the phosphorylated form) was not released by ATP alone; its release required the addition of the cofactor GroES together with ATP. Thus, the phosphorylation of GroEL during heat shock greatly enhances its ability to bind to certain denatured proteins and stimulates its ATP-dependent dissociation in the absence of GroES. Presumably, these alterations in the properties of a fraction of GroEL aid in the refolding or the degradation of specific damaged polypeptides.