We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli 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 thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (</=30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi-chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order-of-addition experiment in which aggregates were formed prior to induction of synthesis of the bi-chaperone 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.
Keywords: chaperones/heat-shock response/Hsp70/protein denaturation/thermotolerance