Abstract

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.