Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

Abstract

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage A DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp7O analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explaln why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.The Escherichia coli dnaK gene product, the prokaryotic analogue of hsp70, the eukaryotic 70-kDa heat shock protein, participates in a variety of basic cellular functions: (i) survival of bacteria under different stress conditions, (ii) initiation of bacteriophage A and E. coli oriC-dependent DNA replication, (iii) regulation of cell division, (iv) modulation of proteolysis, (v) protein phosphorylation, and (vi) transport of proteins across membranes (reviewed in refs. 1-3). Such a broad spectrum of action suggests involvement of the DnaK protein in some general mechanisms crucial for the survival of the cell. Pelham (4) has suggested that the heat shock proteins belonging to the hsp70 family are involved in binding to the hydrophobic domains of other proteins, exposed either naturally or as a result of stressful conditions. Such binding and release, following ATP hydrolysis, may allow the disassembly of "dead-end" protein structures formed under stress conditions. In support of this hypothesis, we have recently shown that the DnaK protein protects E. coli RNA polymerase from heat inactivation by preventing its aggregation. In addition, in an ATP-dependent reaction, the DnaK protein can also dissolve the RNA polymerase aggregates formed at high temperature, leading to a complete restoration of RNA polymerase activity (5). Early evidence that ATP may be involved in hsp70 function was the observation that the E. coli DnaK protein has a weak ATPase activity (6). It was subsequently shown that members of the mammalian hsp70 family of proteins bind tightly to ATP cross-linked to an agarose matrix (7) and that ATP is required for release of hsp70 protein from nuclei (8). Similar results were obtained when ATP was added to complexes of hsc70 (a constitutive member of the hsp70 family) and p53 (an anti-oncogenic protein) (9),...

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

“…4). A wide range of values have been reported for the basal ATPase activity of EcDnaK with V max values ranging from 0.43 to 3.5 nmol Pi/min and K M values ranging from 20 nM to 20 μM ATP [6,40,41].…”

“…The molecular chaperone function of Hsp70/DnaK proteins seems to be based on its ATPase activity and this activity is stimulated by Hsp40/ DnaJ, GrpE and substrate binding [6]. The chaperone activity of Hsp70/DnaK proteins is dependent on a close interaction between the substrate-binding domain and the ATPase domain.…”

“…The ATPase activity is regulated by the co-chaperone protein DnaJ [7,8]. The DnaJ proteins stimulate the low basal ATP hydrolysis activity of partner Hsp70s/DnaKs [6,9] and in doing so enhance the Hsp70s/Dnaks substrate-binding activity and hence their chaperone activity.…”

The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

“…Under physiological conditions of high ATP concentration, nucleotide exchange is rate limiting for substrate release. Nucleotide exchange of DnaK, however, is slow but stimulated 5000-fold by the nucleotide exchange factor GrpE (Liberek et al 1991;Packschies et al 1997;Brehmer et al 2004). The currently accepted mechanism for DnaK-assisted refolding of a denatured protein assumes that a misfolded protein substrate (e.g.…”

Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress‐inducible nucleotide‐exchange factor of ubiquitous appearance in mammalian organs

“…The high conservation of Hsp70 homologues throughout evolution is thought to help maintain their function as molecular chaperones in such diverse cellular reactions as protein folding, hormone receptor function, proteolysis and protein translocation. Central to the function of Hsp70 homologues as molecular chaperones is a weak ATPase activity which at least in the case of DnaK is accelerated about 50-fold by the concerted effort of the heat shock proteins DnaJ and GrpE [3]. Furthermore, DnaJ and GrpE appear to regulate the ability of DnaK to bind unfolded proteins and release them in an ATP-dependent manner and together they comprise a 'chaperone machine' which, in one form or the other, may operate in all compartments of eukaryotic cells.…”