Heat shock transcription factor σ32 co-opts the signal recognition particle to regulate protein homeostasis in E. coli.

All cells must adapt to rapidly changing conditions. The heat shock response (HSR) is an intracellular signaling pathway that maintains proteostasis (protein folding homeostasis), a process critical for survival in all organisms exposed to heat stress or other conditions that alter the folding of the proteome. Yet despite decades of study, the circuitry described for responding to altered protein status in the best-studied bacterium, E. coli, does not faithfully recapitulate the range of cellular responses in response to this stress. Here, we report the discovery of the missing link. Surprisingly, we found that σ(32), the central transcription factor driving the HSR, must be localized to the membrane rather than dispersed in the cytoplasm as previously assumed. Genetic analyses indicate that σ(32) localization results from a protein targeting reaction facilitated by the signal recognition particle (SRP) and its receptor (SR), which together comprise a conserved protein targeting machine and mediate the cotranslational targeting of inner membrane proteins to the membrane. SRP interacts with σ(32) directly and transports it to the inner membrane. Our results show that σ(32) must be membrane-associated to be properly regulated in response to the protein folding status in the cell, explaining how the HSR integrates information from both the cytoplasm and bacterial cell membrane.

Regulation of the heat shock response in Escherichia coli: history and perspectives.

The heat shock response mediated by transcription factor σ32 is a major stress response to cope with heat and other stresses in Escherichia coli. Although much attention has been paid to the role of highly conserved heat shock proteins such as chaperones and proteases in sustaining cellular protein homeostasis under stress, relatively little is known about the dynamic nature of underlying regulatory mechanisms. When cells are suddenly exposed to high temperature, synthesis of σ32 is rapidly induced by activated translation of rpoH mRNA, which encodes σ32, through disruption of mRNA secondary structure. The increased synthesis of σ32 is accompanied by stabilization of σ32, which is normally very unstable and rapidly degraded by the membrane-localized FtsH protease. It was recently found that σ32 must be localized to the inner membrane by the SRP-dependent pathway to work properly for regulation, but the roles played by membrane and other components of the cell remained unknown. Random transposon mutagenesis of the strongly deregulated I54N-σ32 mutant has now started to unravel the complex regulatory circuit, involving membrane protein(s), other cellular components or σ32-interfering polypeptides, for dynamic fine-tuning of σ32 activity that could be of vital importance for cell survival.

Heat shock transcription factor σ32 defective in membrane transport can be suppressed by transposon insertion into genes encoding a restriction enzyme subunit or a putative autotransporter in Escherichia coli.

Heat shock transcription factor σ32 of Escherichia coli plays a major role in protein homeostasis and requires membrane localization for regulation. We here report that a strongly deregulated I54N-σ32 mutant defective in association with the membrane can be phenotypically suppressed by Tn5 insertion into the mcrC or ydbA2 gene, encoding a restriction enzyme subunit or part of a putative autotransporter, respectively. The suppression is specific for mutant I54N-σ32 and reduces its activity but not its abundance or stability. Moreover, the deregulated phenotype of I54N-σ32 is effectively suppressed by a plasmid carrying the same mcrC::Tn5 mutation. In contrast, deletion of the mcrC or ydbA2 gene hardly affects I54N-σ32 activity. These results, taken together, suggest that the truncated form of McrC (and presumably also of YdbA2) protein produced by the Tn5 insertion interacts specifically with I54N-σ32 to reduce its activity without substantially affecting its amount or stability.

A Novel SRP Recognition Sequence in the Homeostatic Control Region of Heat Shock Transcription Factor σ32.

Heat shock response (HSR) generally plays a major role in sustaining protein homeostasis. In Escherichia coli, the activity and amount of the dedicated transcription factor σ(32) transiently increase upon heat shock. The initial induction is followed by chaperone-mediated negative feedback to inactivate and degrade σ(32). Previous work reported that signal recognition particle (SRP)-dependent targeting of σ(32) to the membrane is essential for feedback control, though how SRP recognizes σ(32) remained unknown. Extensive photo- and disulfide cross-linking studies in vivo now reveal that the highly conserved regulatory region of σ(32) that lacks a consecutive hydrophobic stretch interacts with the signal peptide-binding site of Ffh (the protein subunit of SRP). Importantly, the σ(32)-Ffh interaction observed was significantly affected by mutations in this region that compromise the feedback regulation, but not by deleting the DnaK/DnaJ chaperones. Homeostatic regulation of HSR thus requires a novel type of SRP recognition mechanism.

Proteome analysis of plant-induced proteins of Agrobacterium tumefaciens.

Abstract A proteome study of Agrobacterium tumefaciens exposed to plant roots demonstrated the existence of a plant-dependent stimulon. This stimulon was induced by exposure to cut roots and consists of at least 30 soluble proteins (pI 4-7), including several proteins whose involvement in agrobacteria-host interactions has not been previously reported. Exposure of the bacteria to tomato roots also resulted in modification of the proteins: Ribosomal Protein L19, GroEL, AttM, and ChvE, indicating the significance of protein modifications in the interactions of agrobacteria with plants.

The increment of anti-ORP150 autoantibody in initial stages of atheroma in high-fat diet fed mice.

Expression of 150 kda oxygen-regulated protein, ORP150, was examined in the atheromatous lesions on aortic valves in high-fat diet fed mice. Immunohistochemical staining revealed that ORP150 was expressed on the surface of plaque and was co-localized with phagocytes bearing Mac-3, a mouse macrophage differentiation antigen. These findings suggest that ORP150 is involved in the development of the atheromatous plaque. Titer of autoantibody against ORP150 was gradually elevated in parallel with the length of period of high-fat diet feeding. These results suggest that the deposition of immunocomplex toward ORP150 antigen is involved in atheromatous plaque progression.

Convergence of molecular, modeling, and systems approaches for an understanding of the Escherichia coli heat shock response.

The heat shock response (HSR) is a homeostatic response that maintains the proper protein-folding environment in the cell. This response is universal, and many of its components are well conserved from bacteria to humans. In this review, we focus on the regulation of one of the most well-characterized HSRs, that of Escherichia coli. We show that even for this simple model organism, we still do not fully understand the central component of heat shock regulation, a chaperone-mediated negative feedback loop. In addition, we review other components that contribute to the regulation of the HSR in E. coli and discuss how these additional components contribute to regulation. Finally, we discuss recent genomic experiments that reveal additional functional aspects of the HSR.

Analysis of sigma32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response.

Protein quality control is accomplished by inducing chaperones and proteases in response to an altered cellular folding state. In Escherichia coli, expression of chaperones and proteases is positively regulated by sigma32. Chaperone-mediated negative feedback control of sigma32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in sigma32 altered in feedback control. Surprisingly, such mutants are resistant to inhibition by both the DnaK/J and GroEL/S chaperones in vivo and also show dramatically increased stability. Further characterization of the most defective mutant revealed that it has almost normal binding to chaperones and RNA polymerase and is competent for chaperone-mediated inactivation in vitro. We suggest that the mutants identify a regulatory step downstream of chaperone binding that is required for both inactivation and degradation of sigma32.

Conserved region 2.1 of Escherichia coli heat shock transcription factor sigma32 is required for modulating both metabolic stability and transcriptional activity.

Escherichia coli heat shock transcription factor sigma32 is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK, DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of sigma32. To gain further insight into the regulation of sigma32 stability, we isolated sigma32 mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues 47 to 55) of region 2.1, a region highly conserved among bacterial sigma factors. The half-lives ranged from about 2-fold to more than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such as DnaK and GroEL, increased in cells producing stable sigma32. Detailed analysis showed that some stable sigma32 mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region 2.1 is required for modulating both metabolic stability and the activity of sigma32. The evidence suggests that sigma32 stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in interactions with the proteolytic machinery, including molecular chaperones.

Heat shock proteome of Agrobacterium tumefaciens: evidence for new control systems.

The regulation of Agrobacterium tumefaciens heat shock genes involves a transcriptional activator (RpoH) and repressor elements (HrcA-CIRCE). Using proteome analysis and mutants in these control elements, we show that the heat shock induction of 32 (out of 56) heat shock proteins is independent of RpoH and HrcA. These results indicate the existence of additional regulatory factors in the A. tumefaciens heat shock response.