Analysis of σ ³² mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response

Abstract

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 32 . Chaperone-mediated negative feedback control of 32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in 32 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 32 .heat shock transcription factor ͉ proteolysis ͉ GroEL ͉ DnaK ͉ stress response T he heat shock response is a major homeostatic mechanism for controlling the state of protein folding and degradation in all cells (1-3). Upon heat stress, a set of highly conserved heat shock proteins (hsps), including chaperones and proteases, is rapidly and transiently induced. Hsps maintain optimal states of protein folding and turnover during normal growth and also minimize cellular damage from stress-induced protein misfolding and aggregation (4, 5). The level of hsps is controlled primarily by heat shock transcription factors that sense the cellular folding environment through negative feedback control mediated by chaperones (6-9). Understanding this mode of regulation is central to our understanding of protein quality control as well as cellular stress responses. Here, we report the determinants required for chaperone regulation of 32 , the heat shock transcription factor in Escherichia coli (10-12).32 regulon members control both the activity and stability of 32 . The DnaK/J/GrpE and GroEL/S chaperone machines each constitute a negative feedback loop that couples 32 activity to cellular protein folding state: overexpression of either chaperone machine decreases 32 activity; conversely, chaperone depletion or overexpression of chaperone substrates increases 32 activity (13-16). The chaperones are likely to act directly on 32 because they bind to 32 and inhibit its activity in a purified in vitro transcription system (17-19). Regulated degradation of 32 is mediated by the FtsH protease and facilitated by DnaK/J/GrpE and GroEL/S in vivo, but this process has not been completely recapitulated in vitro, where degradation of 32 by FtsH is slow and not facilitated by chaperones (20,21).We selected and characterized feedback-resistant mutants of 32 . The residues altered by the mutations map to a small patch in 32 . Surprisingly, most mutants simultaneously diminished all three negative feedback loops operative in vivo; the most severe mutant essentially eliminated chaperone-mediated activity control and degradation by FtsH protease. In stri...

Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH

“…E. coli K-12 strain MG1655 was transformed with either pRB11 (Yura et al, 2007) or pBADrpoH. Phage P1 was used to transduce the DrpoH : : Kan zhf50 : : Tn10 mutation from strain CAG48315 (Zhou et al, 1988) into MG1655/ pRB11 and MG1655/pBADrpoH.…”

GroEL to DnaK chaperone network behind the stability modulation of σ32 at physiological temperature in Escherichia coli

“…Although there were reports concerning in vitro interactions between r 32 and DnaK/GroEL, an in vivo interaction was evident only for DnaK [12,31,32]. Here we show for the first time the evidence for in vivo physical association between r 32 and GroEL, by coimmunoprecipitation experiments.…”

“…1A and Fig. S1) implied that in the immunoprecipitate, both GroEL and DnaK were bound to r 32 , forming either a ternary complex GroEL-r 32 -DnaK or binary complexes GroEL-r 32 and DnaK-r 32 . To investigate the exact Co-immunoprecipitation of native cell lysate was done with anti-r 32 antibody and subsequent western blot was done using anti-r 32 antibody and (anti-GroEL + antiDnaK) antibody mix separately with lysates of MC4100 cells at 30°C and of MC4100 DrpoH cells at 20°C.…”

“…Therefore, Figs. 3 and 4 signified that the amounts of total precipitated r 32 and DnaK in groEL mutant cells were about two fold higher than the amounts in (wt) cells. This result further indicated conformity with the 'unfolded protein titration model' and so, the amounts of both r 32 and DnaK were greater in groEL mutant cells than in (wt) cells (Fig.…”

“…Escherichia coli heat-shock genes for chaperones and proteases are transcribed by RNA polymerase with the alternative sigma factor r 32 . The cellular level of r 32 (10-30 copies/cell) increases by 12-15-fold upon shifting the cells from physiological temperature (30°C) to a heat-stress one (45°C), with transient induction of chaperones and proteases [7][8][9][10][11].…”