The cellular stress response is a universal mechanism of extraordinary physiological/pathophysiological significance. It represents a defense reaction of cells to damage that environmental forces inflict on macromolecules. Many aspects of the cellular stress response are not stressor specific because cells monitor stress based on macromolecular damage without regard to the type of stress that causes such damage. Cellular mechanisms activated by DNA damage and protein damage are interconnected and share common elements. Other cellular responses directed at re-establishing homeostasis are stressor specific and often activated in parallel to the cellular stress response. All organisms have stress proteins, and universally conserved stress proteins can be regarded as the minimal stress proteome. Functional analysis of the minimal stress proteome yields information about key aspects of the cellular stress response, including physiological mechanisms of sensing membrane lipid, protein, and DNA damage; redox sensing and regulation; cell cycle control; macromolecular stabilization/repair; and control of energy metabolism. In addition, cells can quantify stress and activate a death program (apoptosis) when tolerance limits are exceeded.

Molecular and evolutionary basis of the cellular stress response

Promoter recognition in eubacteria is carried out by the initiation factor sigma, which binds RNA polymerase and initiates transcription. Cells have one housekeeping factor and a variable number of alternative sigma factors that possess different promoter-recognition properties. The cell can choose from its repertoire of sigmas to alter its transcriptional program in response to stress. Recent structural information illuminates the process of initiation and also shows that the two key sigma domains are structurally conserved, even among diverse family members. We use the sigma repertoire of Escherichia coli, Bacillus subtilis, Streptomyces coelicolor, and cyanobacteria to illustrate the different strategies utilized to organize transcriptional space using multiple sigma factors.

Multiple sigma subunits and the partitioning of bacterial transcription space.

Certain proteins and protein fragments in Escherichia coli are modified by carboxy-terminal addition of an 11-residue peptide tag (Tu et al. 1995). This tagging process requires functional SsrA RNA (10Sa RNA), which encodes the last 10 residues of the peptide (Tu et al. 1995) and results in rapid degradation of the tagged protein by carboxy-terminal-specific proteases (Keiler et al. 1996). SsrA-mediated tagging of proteins translated from defective messenger RNAs lacking termination codons has been demonstrated, and a model in which SsrA functions both as a tRNA and an mRNA has been proposed (Keiler et al. 1996). The 363-nucleotide SsrA RNA has sequences that form a tRNA-like structure and has been shown to be chargeable with alanine (Komine et al. 1994; Williams and Bartel 1996; Felden et al. 1997). In the model proposed by Keiler et al., when ribosomes stall at the 3′ end of the damaged message, SsrA charged with alanine binds to the ribosome like a tRNA and contributes the alanine to the idle nascent chain. Translation then switches from the mRNA to a small open reading frame (ORF) in SsrA that encodes the carboxy-terminal degradation peptide. This system provides both a method to avoid the accumulation of ribosomes stalled at the end of defective messages and a general quality-control mechanism that allows the cell to rid itself of incomplete protein fragments that might have inappropriate cellular activities. Cells devoid of SsrA RNA grow more slowly and show a certain degree of temperature sensitivity (Oh and Apirion 1991; Komine et al. 1994; Trempy et al. 1994).

The involvement of carboxy-terminal amino-acid sequences in targeting proteins for rapid degradation was recognized before the discovery of the SsrA-tagging system (Bowie and Sauer 1989; Parsell et al. 1990), and a periplasmic protease (Tsp or Prc) that degrades protein substrates in a carboxy-terminal-specific manner was purified and characterized (Silber et al. 1992). The carboxy-terminal substrate sequences recognized by Tsp are similar to those of the SsrA tag (Keiler et al. 1995; Tu et al. 1995), and Tsp is responsible for degradation of SsrA-tagged proteins that are exported to the periplasm (Keiler et al. 1996). Cytoplasmic proteins with carboxy-terminal degradation sequences, however, are still proteolyzed rapidly in cells lacking Tsp (Silber and Sauer 1994; Keiler et al. 1996), indicating that other proteases must be responsible for carboxy-terminal-specific degradation of proteins in the bacterial cytoplasm.

Essentially all cytoplasmic degradation in prokaryotes, archaea, and eukaryotes is energy-dependent. E. coli, for example, has at least five ATP-dependent proteases [Lon (La); HflB (FtsH); ClpAP; ClpXP; and ClpYQ (HslUV)] (for review, see Gottesman 1996). These enzymes appear to have distinct substrate preferences, as a mutation in a single protease gene is often sufficient to stabilize a specific unstable protein. For example, mutations in Lon lead to stabilization of the N protein of bacteriophage λ, the SulA and RcsA proteins of E. coli, and the CcdA protein of the episomal F factor. HflB appears to be responsible for degradation of the cII protein of λ and the heat-shock σ factor RpoH (Herman et al. 1993, 1995). The principal substrates for ClpYQ degradation have not yet been identified, although this two-component protease has been implicated in degradation of both Lon subtsrates and HflB substrates in vivo (Missiakas et al. 1996; Kanemori et al. 1997; Khattar 1997; W.-F. Wu and S. Gottesman, unpubl.). ClpAP and ClpXP are two-component proteases that share a common proteolytic subunit, ClpP, but have different ATPase regulatory subunits, ClpA or ClpX. Proteins stabilized by mutations in clpX but not in clpA include λ O, phage Mu repressor variants, and the stationary-phase σ factor, RpoS; clpA but not clpX mutants stabilize certain LacZ fusion proteins and the MazE protein. ClpB, an ATPase with extensive sequence similarity to ClpA, has not thus far been demonstrated to have a direct role in proteolysis but may act as a chaperone (Squires and Squires 1992).

In the studies presented here, we show that intracellular degradation of variants of the amino-terminal domain of λ repressor containing the SsrA peptide tag is dramatically reduced in cells lacking ClpP or lacking both ClpX and ClpA, and is somewhat reduced in cells lacking ClpX or ClpA only. Purified ClpXP and purified ClpAP degrade SsrA-tagged protein substrates in vitro, suggesting that these ATP-dependent enzymes are directly responsible for degradation of SsrA-tagged proteins in the bacterial cytoplasm.

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The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system

Evolutionary history and higher order classification of AAA+ ATPases.

Proteolysis in Escherichia coli serves to rid the cell of abnormal and misfolded proteins and to limit the time and amounts of availability of critical regulatory proteins. Most intracellular proteolysis is initiated by energy-dependent proteases, including Lon, ClpXP, and HflB; HflB is the only essential E. coli protease. The ATPase domains of these proteases mediate substrate recognition. Recognition elements in target are not well defined, but are probably not specific amino acid sequences. Naturally unstable protein substrates include the regulatory sigma factors for heat shock and stationary phase gene expression, sigma 32 and RpoS. Other cellular proteins serve as environmental sensors that modulate the availability of the unstable proteins to the proteases, resulting in rapid changes in sigma factor levels and therefore in gene transcription. Many of the specific proteases found in E. coli are well-conserved in both prokaryotes and eukaryotes, and serve critical functions in developmental systems.

Proteases and their targets in Escherichia coli.

Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1995.tb07253.x

J. Gamer

Papers

Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.