Integrating protein homeostasis strategies in prokaryotes

Abstract

Bacterial cells are frequently exposed to dramatic fluctuations in their environment, which cause perturbation in protein homeostasis and lead to protein misfolding. Bacteria have therefore evolved powerful quality control networks consisting of chaperones and proteases that cooperate to monitor the folding states of proteins and to remove misfolded conformers through either refolding or degradation. The levels of the quality control components are adjusted to the folding state of the cellular proteome through the induction of compartment specific stress responses. In addition, the activities of several quality control components are directly controlled by these stresses, allowing for fast activation. Severe stress can, however, overcome the protective function of the proteostasis network leading to the formation of protein aggregates, which are sequestered at the cell poles. Protein aggregates are either solubilized by AAA+ chaperones or eliminated through cell division, allowing for the generation of damage-free daughter cells.

Figure 1.

Interplay of chaperone system during de novo protein folding in E. coli. Nascent polypeptides initially interact with ribosome-bound Trigger Factor (TF), which binds to the ribosomal protein L23. On release from TF, newly synthesized proteins either fold spontaneously (roughly estimated two thirds of cytosolic proteins under physiological growth conditions) or require further folding-assistance by downstream chaperones, namely the Hsp70 chaperone DnaK, which acts together with its co-chaperone DnaJ and the nucleotide exchange factor GrpE, and/or the Hsp60 chaperone GroEL with its co-chaperone GroES. The ATP-dependent DnaK- and GroEL-machineries may act co- and/or posttranslationally.

Figure 2.

Regulation of bacterial stress responses. (A) Principle of RNA thermometers. At low temperatures, the ribosomal binding site (RBS) and the AUG start codon of an mRNA encoding for a stress gene is base paired and not accessible. On heat shock, the structure around the RBS melts allowing for ribosome binding (30S and 50S) and translation. (B) Chaperones and proteases link the cellular folding state to stress gene expression. Under nonstress conditions, expression of stress genes is inhibited through (1) inhibition of transcriptional activators by chaperones or proteases that either sequester the regulators or degrade them or (2) repressor proteins that require chaperone assistance for activity. During environmental stress misfolded proteins accumulate, which titrate chaperones and proteases from their regulatory roles and resulting in the expression of stress genes through (1) release or stabilization of transcriptional activators or (2) inactivation of repressor proteins. Expression of stress genes initiates an inactivation feedback loop restoring nonstress gene expression.

Figure 3.

Activities of bacterial quality control systems during environmental stress. Environmental stress like heat shock can cause protein-unfolding leading to the accumulation of misfolded protein species. Misfolded proteins are either refolded by the DnaK chaperone and its co-chaperone DnaJ or are removed by AAA+ proteases including e.g., Lon, ClpC/ClpP or HslU/HslV. The holding chaperone Hsp33 becomes important under oxidative and thermal stress and prevents protein aggregation. Severe stress conditions can overburden the protective capacity of quality control systems causing protein aggregation. sHsps coaggregate with misfolded protein species thereby changing the architecture (physical properties) of aggregates and allowing for more efficient protein disaggregation by chaperones. DnaK/DnaJ in cooperation with the AAA+ chaperone ClpB efficiently solubilize protein aggregates by extracting single unfolded protein species, whereas DnaK/DnaJ alone have limited disaggregation capacity. AAA+ proteases (ClpC/ClpP) might also act on aggregated protein species.

Figure 4.

Sequestration of protein aggregates at polar sites allows for aggregate clearance by cell division. In E. coli misfolded proteins are deposited during stress conditions as inclusion bodies at polar sites. The sequestration at these sites is driven by nucleoid occlusion resulting in the accumulation of misfolded protein species or small aggregates at the nucleoid-free space. The formation of a single protein aggregate, which is preferentially deposited at the old cell pole, allows for the asymmetric inheritance of aggregated proteins through cell division. New pole cells that are damage-free show higher growth rates at the expense of old pole cells, which retain the damage.

Figure 5.

Periplasmic quality control system. In the cytosol secretory proteins are kept in an export-competent conformation through association with the chaperone SecB. On delivery to SecA, the secretory proteins are translocated through the SecYEG translocon into the periplasm. Folding of newly imported proteins is supported by periplasmic chaperones including Skp, FkpB or SurA. Oxidative folding (formation of disulfide bonds) is catalyzed by DsbA, whereas incorrect disulfides are isomerized by DsbC/DsbG. Oxidized DsbA and DsbC/G are regenerated by DsbB and DsbD, respectively. Misfolded proteins are degraded by DegP. High levels of misfolded protein species can cause the generation of protein aggregates that might be removed by DegP.