Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70–Hsp40 Substrates

Abstract

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70–Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease.

The Hsp70-Chaperone Machines in Bacteria

“…According to a recent quantitative proteomics study, DnaK constitutes under non-stress conditions 98% (ca. 34 µM) of all Hsp70 proteins and HscA 2% (0.8 µM), whereas HscC was below the detection limit (Fauvet et al, 2021). <0.1 indicates that these components were below the detection limit of this quantitative mass spectrometry experiment.…”

“…(<0.1 µM) (Fauvet et al, 2021) (Figure 8A). GrpE (ca 18 µM) is about half as abundant as DnaK resulting in a stoichiometry of one GrpE dimer per four DnaK molecules.…”

“…According to a recent quantitative proteomics study, DnaK constitutes under non-stress conditions 98% (ca. 34 µM) of all Hsp70 proteins and HscA 2% (0.8 µM), whereas HscC was below the detection limit (<0.1 µM) ( Fauvet et al, 2021 ) ( Figure 8A ). GrpE (ca 18 µM) is about half as abundant as DnaK resulting in a stoichiometry of one GrpE dimer per four DnaK molecules.…”

“…(A) , Concentrations of the components of the Hsp70 systems in wild type E. coli in non-stress conditions and exponential growth phase. Numbers indicate concentrations in µM according to ( Fauvet et al, 2021 ). <0.1 indicates that these components were below the detection limit of this quantitative mass spectrometry experiment.…”

Repair or Degrade: the Thermodynamic Dilemma of Cellular Protein Quality-Control

“…As mentioned, in E. coli, the protein repair part of the proteostasis network is mostly composed of the conserved chaperone families Hsp70s (DnaK, HscA), Hsp60s (GroEL), Hsp90s (HtpG), Hsp100 (ClpB) and Hsp20s (IbpA/B) (bacterial names in brackets), with their main co-chaperones (DnaJ, CbpA, DjlA, HscB, GrpE, GroES) (Finka and Goloubinoff, 2013). Indicating the importance of this chaperone network, its members contribute ∼3.3% of the total proteome mass of unstressed E. coli cells, compared to ∼1% for the endo-cellular proteases, with ∼0.6% of the total proteome belonging to qualitycontrol proteases (Figure 2A), (Fauvet et al, 2021).…”