The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli

Abstract

Highlights d Acute heat stress causes unfolding of a thermo-sensitive subproteome in E. coli d DnaK (Hsp70) markedly stabilizes most thermo-sensitive proteins d Numerous essential ribosomal proteins are conformationally stabilized by DnaK d Other DnaK-stabilized proteins tend to be large, multidomain, and hetero-oligomeric SUMMARYStress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify $1,500 soluble proteins and find $500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional $200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.

Escherichia colismall heat shock protein IbpA is an aggregation-sensor that self-regulates its own expression at a translational level

“…The addition of copper disturbs protein homeostasis in cells with oxidative stress [24,26]. A common consequence in E. coli cells would be the accumulation of protein aggregates [20][21][22][23]. Therefore, we hypothesized that protein aggregation might somehow be involved in the upregulation of IbpA under the non-heat stressed conditions.…”

“…Although the thermometer in the mRNA (RNAT) and the transcriptional control by σ 32 are known mechanisms to upregulate the expression of IbpA, previous studies have reported that IbpA expression is also upregulated under non-heat stressed conditions, such as in the dnaKJ deletion strain or upon copper stress [22][23][24]. The absence of DnaK/DnaJ leads to the production of protein aggregates [22,25].…”

“…Previous analyses of the RNAT in ibpB using a reporter revealed the possible influence of the IbpA protein on ibpB expression [7,21]. In addition to the heat stress, the expression level of IbpA/IbpB was profoundly upregulated, by 10~50-fold, under non-heat stressed conditions such as 30~37 °C in the dnaK-dnaJ deleted strain [22,23] or upon oxidative stress induced by copper [24]. Since the RNAT regulation would not be effective at normal growth temperatures, the mechanism for the massive upregulation under the non-heat stressed conditions remains to be elucidated.…”

Programmed trade-offs in protein folding networks

“…The prevalence of structural homologues that differ in their chaperone dependence clearly indicates that the protein fold alone is in general not a stringent requirement for chaperone interaction (18,26,32). Comparative analyses of homologous proteins thus allow to delineate any trade-offs within alternate sequences that fold into the same native protein structures.…”

“…Understanding the determinants of specificity and selectivity of ribosomeassociated chaperones is of particular interest as they are directly coupled to the sequence de-terminants of de novo protein folding in the cell (21,31). Importantly, the integration of protein structures into chaperone interaction maps has highlighted that the structural fold of proteins alone is generally not a determining factor of chaperone interaction (18,26,32). Thus, through tremendous progress on understanding chaperone action and interaction, it has also become increasingly clear that not a single feature but rather the complex interplay of multiple protein characteristics determines their interaction with chaperones.…”