Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coil to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of t hermolabile proteins, a surprisingly high number of 150-200 species, corres ponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the lar ge (greater than or equal to 90 kDa) but only 18% of the small (less than o r equal to 30 kDa) cytosolic proteins and include essential proteins, The D naK system in addition acts with ClpB to form a bi-chaperone system that qu antitatively solubilizes aggregates of most of these proteins. Efficient so lubilization also occurred in an in vivo order-of-addition experiment in wh ich aggregates were formed prior to induction of synthesis of the bi-chaper one system. Our data indicate that large-sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central , dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation.