Open AccessThe effect of co‐overproduction of DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat‐aggregated proteins from Escherichia coli Δ clpB mutant cells – new insight into the role of Hsp70 in a functional cooperation with Hsp100
Author(s) -
Kedzierska Sabina,
Matuszewska Ewelina
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10910.x
Subject(s) - clpb , escherichia coli , overproduction , heat shock protein , mutant , biology , plasmid , wild type , biochemistry , hsp70 , heat shock , microbiology and biotechnology , chaperone (clinical) , gene , medicine , pathology
The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat‐aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were 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 (σ 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°C. 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 remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat‐aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5‐fold that of wild‐type) in wild‐type and Δ clpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight‐fold that of wild‐type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co‐production of DnaK/DnaJ/GrpE and ClpB in the Δ clpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat‐shocked E. coli cells. In the presence of excess ClpB, an increase 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, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.