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

Abstract

Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.

Firefly luciferase mutants as sensors of proteome stress

“…Quantitative immunoprecipitation analysis in mammalian cells demonstrated that Hsc70 transiently and preferentially associates with elongating polypeptides larger than 20 kDa and at least 15-20% of newly synthesized proteins associate with Hsc70 during their biogenesis (Thulasiraman et al, 1999). These results are consistent with findings in prokaryotes where DnaK was shown to interact with ~ 15% of polypeptides and has been shown to facilitate the post-translational folding of multi-domain proteins through several cycles of binding and release (Calloni, 2012;Teter et al, 1999).…”

“…For instance, it was demonstrated in E. coli that deletion of DnaK in TF-deleted cells resulted in massive aggregation of cytosolic proteins and combined deletion of both TF and DnaK caused synthetic lethality (Calloni, 2012;Deuerling et al, 1999). Further, upon deletion of TF, the polypeptide flux through DnaK increases from ~ 15% to ~ 40% showing that there is partial functional redundancy between different classes of chaperones (Teter et al, 1999).…”

In vivo translation rates can substantially delay the cotranslational folding of the Escherichia coli cytosolic proteome

“…Trigger factor (TF) and DnaK are two cotranslationally acting E. coli chaperones that preferentially bind to the nascent chains of larger proteins compared with smaller ones (38,39). In E. coli cells in which DnaK and TF were deleted, it was found that larger proteins were much more likely to aggregate than smaller proteins (38)(39)(40).…”

GroEL to DnaK chaperone network behind the stability modulation of σ32 at physiological temperature in Escherichia coli

“…For native folding of about 10% E. coli proteins, where DnaKGroE systems work together, the DnaK system was known to act upstream of the GroE system [18][19][20][36][37]. This fact led us to investigate whether the same sequence of action existed for in vivo modulation of r 32 stability at 30°C.…”

“…According to the current knowledge, for attainment of an active conformation, 18-20% cytosolic proteins require assistance of the DnaK system, 6-8% proteins require the GroE system and about 10% proteins require both the DnaK and GroE systems where GroEL acts downstream of DnaK [17][18][19][20]. Author contributions: MP contributed substantially in designing and performing the experiments.…”