DnaK Functions as a Central Hub in the E. coli Chaperone Network

Upon emerging from the ribosomal exit tunnel, folding of the
polypeptide chain is necessary to form the fully functional
protein. In E. coli, correct and efficient protein folding is
mainly secured by an organized and complex chaperone system which
includes two main principles: The first principle consists of the
nascent binding chaperones including trigger factor (TF) and the
DnaK/DnaJ system, while the second principle is represented by the
downstream GroEL/ES chaperonin system. The identification of ~250
natural GroEL substrates demonstrated that GroEL/ES specifically
folds a small group of proteins with complex domain topologies
(Kerner et al., 2005) which include some essential proteins.
Although the structural, functional and mechanistic aspects of
DnaK, the E. coli Hsp70 chaperone, have been extensively studied, a
systematic profiling of the natural DnaK substrates is still
missing. Moreover, the cooperation between the two main chaperone
systems remains to be elucidated. Here we analyzed the central role
of DnaK in the bacterial chaperone network and its cooperation with
the ribosome-associated chaperone TF and the downstream chaperonin
GroEL/GroES using SILAC-based proteomics of DnaK-pulldowns. In
parallel, we also analyzed the changes at the global proteome level
under conditions of single or combined chaperone deletion. Our
measurements show that DnaK normally interacts with at least ~700
newly-synthesized and pre-existent proteins (~30 % of all cytosolic
proteins), including ~200 aggregation-prone substrates. Individual
deletion of TF or depletion of GroEL/ES at 30 oC-37 oC leads to
limited but highly specific changes in the DnaK interactome and in
global proteome composition. Specifically, loss of TF results in
increased interaction of DnaK with ribosomal and other small, basic
proteins, and in a specific defect in the biogenesis of outer
membrane -barrel proteins. While deletion of DnaK/DnaJ leads to
the degradation or aggregation of ~150 highly DnaK-dependent
proteins of large size, massive proteostasis collapse is only
observed upon combined deletion of the DnaK system and TF, and is
accompanied by extensive aggregation of GroEL substrates and
ribosomal proteins. We conclude that DnaK is a central hub in the
cytosolic E. coli chaperone network, interfacing with the upstream
TF and the downstream chaperonin. These three major chaperone
machineries have partially overlapping and non-redundant functions.