The effect of co-overproduction of DnaK/DnaJ/GrpE and ClpB proteins on theremoval of heat-aggregated proteins from Escherichia coli Delta clpB mutant cells - new insight into the role of Hsp70 in a functional cooperation with Hsp100

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE ) and/or ClpB (Hsp100) from plasmids on the process of formation and remova l of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids w ere employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma (32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degreesC. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins rem ained stable 30 min after heat shock, This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproducti on of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-t ype) in wild-type and Delta clpB cells completely prevented the formation o f the S fraction during heat shock. Overproduction of ClpB (exceeding by ab out eight-fold that of wild-type) in the same background did not prevent pr otein aggregation after heat shock and only partly compensated for the effe ct of the mutation in the c[PB gene. Monitoring the S fraction during co-pr oduction of DnaK/DnaJ/GrpE and ClpB in the Delta clpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system protei ns had a significant effect on the formation and removal of protein aggrega tes in heat-shocked E. coli cells. In the presence of excess ClpB, an incre ase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, ma y support protein aggregation resulting from heat shock and may lead to sta bilization of hydrophobic aggregates. (C) 2001 Federation of European Micro biological Societies. Published by Elsevier Science B.V. All rights reserve d.