DnaK protein

Wickner and colleagues have demonstrated a role for the E. coli dnaK protein in replication of bacteriophage PI (Wickner, 1990). In PI replication, the phage repA protein binds specifically to the PI origin of replication it appears to be a monomeric repA that binds to the origin with high affinity. Dimers of repA, in a 2 2 subunit complex with the E. coli... 

Hence, several in vitro activities have demonstrated a participation of stress-70 proteins in the disassembly of macromolecular complexes. The participation of dnaK in initiation of replication for X and PI phages appears to be dependent on ancillary proteins, specifically dnaJ and grpE. This raises the possibility that substrate specificity, in these cases, may be intrinsic to the ancillary proteins rather than residing solely in the dnaK protein. In the case of in vitro dissassembly of clathrin cages, the HSC70 protein can accomplish the reaction without accessory proteins. In all the above cases, ATP hydrolysis is essential for the activities. 

Under certain conditions, the stress-70 proteins can participate in the renaturation of denatured or inactivated proteins. The renaturation capabilities of E. coli dnaK protein have been most extensively documented. It has been shown that in vitro, dnaK can protect E. coli RNA polymerase from aggregation when the polymerase is incubated at elevated temperatures that would normally result in loss of activity, and, further, that dnaK can disaggregate and reactivate polymerase, once it has been inactivated by heat denaturation (Skowyra et al., 1990). These activities are absolutely dependent on ATP hydrolysis. The mutant dnaK756 protein is effective in protecting active RNA polymerase against heat inactivation, but is incapable of disaggregating and reactivating polymerase, once it has been heat inactivated. 

Rose et al., 1989) as well as in the plant Nicotiana tabacum (Denecke et al., 1991), and DDEL in Kluyveromyces lactis (Lewis and Pelham, 1990). The dnaK proteins have a 10- to 20-residue G,A,Q-rich segment, which leads into a highly charged, predominantly acidic termination. In this case, no specialized function has been ascribed to the carboxy-terminal sequence motif. 

The turnover rate of ATP by stress-70 proteins is relatively slow, and is enhanced severalfold by the binding of peptides or by the action of accessory proteins. In the absence of peptides, the E. coli dnaK protein has been reported to have basal ATPase rates in the range of 0.15 (mol ATP/mol dnaK min measured at 37°C, pH 8.1) (McCarty and Walker, 1991) to 0.21 (mol ATP/mol dnaK min measured at 30°C, pH 8.8) (Liberek et ai, 1991a). The basal ATPase rate of dnaK rises steeply as a function of temperature, increasing 70-fold from 20 to 53°C. Above 53°C, the ATPase activity drops precipitously, possibly due to the onset... 

The specific effects of purified dnaj and grpE proteins on the dnaK protein have been characterized (Liberek et al., 1991a). The dnaj and grpE proteins, acting in concert, can stimulate the basal ATPase activity of dnaK 50-fold dnaj appears to enhance the rate of nucleotide hydrolysis by dnaK, but does not substantially affect nucleotide release. The grpE protein appears to enhance the rate of release of nucleotide bound to dnaK. Consequently, neither protein alone exerts a dramatic effect on the overall rate of ATP turnover by dnaK however, the effect of the two proteins combined is to accelerate both the rate of ATP hydrolysis and the rate of nucleotide release, resulting in enhanced ATP turnover. The concentrations of dnaj and grpE required for half-maximal effect are both 0.1-0.2 fiM. 

Kornak.J., Kuo, C., and Campbell, L. (1991). Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein homolog. Infect. Immun. 59, Ti —T2.b. 

RieuI, C., Cortay, J. C., Bleicher, F., and Cozzone, A. J. (1987). Effect of bacteriophage M13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins. Eur.J. Biochem. 168, 621-627. 

Clarke, C.F., Cheng, K., Frey, A.B., Stein, R., Hinds, P.W.. Levine, A.J. (1988). Purification of complexes of nuclear oncogene p53 with rat and E. coli heat shock protein In vitro dissociation of hsc70 and dnaK from murine p53 by ATP. Mol. Cell. Biol. 8, 1206-1215. 

Gamer, J., Bujard, H., Bukua, B. (1992). Physical interaction between heat shock proteins DnaK, DnaJ, CJtpE the bacterial heat shock transcription factor sigma 32. Cell 69, 833-842. 

Langer, T., Lu., C, Echols, H., Flanagan, J, Hayer, M.K., Hartl, F.-U. (1992). Successive action of Dnak, DnaJ, GroEL along the pathway of chaperone mediated protein folding. Nature 356, 683-689. 

It is generally believed that energy-independent chaperones are unable to allow refolding of non-native proteins. They bind to hydrophobic surfaces of denatured proteins to prevent aggregation and pass them to energy-dependent chaperones such as the DnaK or the GroE chaperone machines. 

Falah M, Gupta RS (1994) Cloning of the hsp70 (dnaK) genes from Rhizobium meliloti and Pseudomonas cepacia phylogenetic analyses of mitochondrial origin based on a highly conserved protein sequence. J Bacteriol 176 7748-7753 Ferro M et al. (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol Cell Proteomics 2 325-345... 

---