Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation.

Abstract

To test the idea that unfolded protein might act as an intracellular signal for induction of the heat shock response in Escbericbia coli, we examined the synthesis of several heat shock proteins after expression of an unfolded variant of the amino-terminal domain of X repressor. These experiments show that expression of a single mutant protein, and not its wild-type counterpart, is sufficient to induce a heat shock-like response. In addition, by measuring the abilities of unfolded variants of differing proteolytic susceptibilities to induce heat shock protein synthesis and by monitoring heat shock protein synthesis as a function of the amount of a single unfolded protein, we show that it is the concentration of unfolded protein in the cell, and not its degradation, that is important for inducing the heat shock-like response.

Bacterial growth inhibition by overproduction of protein

“…It might be tempting to look for a role for the heat-shock proteins in this cascade of events, because they are demonstrably augmented in cells that are producing large quantities of a gratuitous protein (Bahl et al, 1987;Parsell and Sauer, 1989;Vind et al, 1993;Dong et al, 1995). Nevertheless, none of the known functions of these proteins provides a ready explanation for how or why ribosomes are selectively destroyed.…”

“…After the initial attempts to make vast quantities of the targeted protein have failed, the experimenters may, in desperation, study their cultures. They then find that the fully induced bacteria are behaving wierdly: they grow slowly (Springer et al, 1985;Franklyn and Schimmel, 1990;Menguito et al, 1993;Tubulekas and Hughes, 1993;Vind et al, 1993;Bowrin et al, 1994), they produce enhanced quantities of heat-shock proteins (Bahl et al, 1987;Parsell and Sauer, 1989;Vind et al, 1993;Dong et al, 1995), and they may eventually stop growing altogether (Dong et al, 1995). Under the light microscope the induced bacteria may look like 'snakes' and they may accumulate inclusion bodies (Marston, 1986).…”

Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

“…Previously, we had also shown that mutated protein Cop∆5, with a half-life of 31p6 min, is moderately less stable than wild-type CopR. Four of the C-terminal 7 aa and two of the C-terminal 5 aa are hydrophilic, which does support the role of hydrophilic amino acids in protecting more hydrophobic regions of the protein against proteolytic attack, as suggested by the publications of R. T. Sauer's group (Milla et al, 1993 ;Parsell & Sauer, 1989 ;Parsell et al, 1990). …”

“…Pioneering work in this field has been done by the group of R. T. Sauer, who investigated sequences stabilizing the Arc repressor and the unstable N terminus of λ repressor (e.g. Bowie & Sauer, 1989 ;Parsell & Sauer, 1989 ;Parsell et al, 1990 ;Milla et al, 1993Milla et al, , 1994. They found that hydrophilic and polar residues within C-terminal extensions can stabilize otherwise unstable proteins, and even classified amino acids depending on their stabilizing effect.…”

Regulation and conservation of the heat‐shock transcription factor σ32

“…The level of unfolded proteins rather than protein degradation is important in enhancing HSP synthesis (Parsell & Sauer 1989). Accumulation of secretory protein precursors, abnormal proteins produced in the presence of azetidine or human prourokinase in E. coli, can all induce HSP synthesis by stabilizing 32 (Wild et al 1993;Kanemori et al 1994) and not by inducing synthesis of 32 (Kanemori et al 1994).…”