![]() We quantitated the amount of DegP on a Western blot using cells grown at 44☌ expressing wild-type degP and degPS210A from pCS20 and pCS21, respectively, induced with 10 and 50 μM IPTG. However, compared to wild-type degP, a higher level of degPS210A expression was required for complementation. Colony formation of degP null mutants expressing degPS210A at 44☌ indicated that the chaperone activity of DegP was sufficient to rescue the cell from thermal folding stress ( Figure 7). If the chaperone activity of DegP is sufficient to reduce folding stress caused by elevated temperatures, the expression of degPS210A should suppress the temperature sensitivity of degP null strains. As expected, DegP S210A did not degrade denatured MalS.Īdditional in vivo evidence for the general molecular chaperone activity of DegP was obtained by investigating the T s phenotype of degP null mutants. At 28☌, incomplete degradation of MalS was observed that was in agreement with the lower protease activity detected when using resorufin-labeled casein as a substrate. As documented for selected samples ( Figure 4B), wild-type DegP degraded MalS at 37☌ and 42☌. Essentially the same results were obtained as for resorufin-labeled casein. ![]() To further investigate the temperature dependence of the proteolytic DegP activity and to verify that DegP S210A is indeed proteolytically inactive, we coincubated denatured MalS and DegP under reducing conditions, in the presence of 30 mM DTT, over the whole temperature range. ![]() This finding explained the temperature-dependent degradation of MalS in the in vitro refolding assays ( Figure 3B). At 28☌, DegP was 8-fold less active than at 42☌, and a major increase in proteolytic activity was observed between 32☌ and 42☌ ( Figure 4A). Below 22☌, DegP was proteolytically inactive. Proteolytic activity was assayed between 4☌ and 42☌, using purified DegP. To obtain quantitative data for the temperature dependence of the proteolytic DegP activity, we used resorufin-labeled casein as a substrate. So far our results have indicated that wild-type DegP has molecular chaperone activity at low temperatures and proteolytic activity at elevated temperatures. The explanation that DegP could first refold and subsequently degrade properly folded MalS can be excluded, since we detected that properly folded MalS was no substrate of DegP (data not shown). At 42☌, the amount of oxidized MalS was the same in samples containing glutathione and wild-type DegP, respectively, suggesting that wild-type DegP did not refold MalS under these conditions. These results indicated that wild-type DegP has chaperone activity at low temperatures and protease activity at elevated temperatures. In the presence of wild-type DegP, degradation of reduced MalS was observed and the total amount of MalS was lower compared to samples containing DegP S210A ( Figure 3B). At 37☌ and 42☌, DegP S210A also stimulated refolding of MalS. The addition of equimolar concentrations of BSA did not stimulate refolding of MalS ( Figure 3A). At 28☌, both DegP and DegP S210A stimulated refolding of MalS more efficiently than glutathione. DegP and DegP S210A were directly added to the refolding assays. We carried out refolding of reduced and denatured MalS in the presence of purified wild-type DegP and the proteolytically inactive DegP S210A mutant ( Figure 3). Refolding Assay of Reduced and Denatured MalS In dsbA degP double null mutants expressing degPS210A, MalS activity was increased 11-fold over the dsbA degP double mutant background ( Table 1). In dsbA degP double null mutants, MalS activity was 30-fold lower as compared to wild-type strains and 18-fold reduced as compared to dsbA mutants. ![]() These results were supported by assaying amylase activity. In this strain, a fraction of MalS could fold into the oxidized and soluble form, since it fractionated with the supernatant of shocked cells. To obtain further evidence for a potential chaperone activity of DegP, we expressed a proteolytically inactive variant of degP ( degPS210A) in dsbA degP double null mutants. These results indicated that DegP may be involved in the folding of MalS in dsbA mutants at low temperatures. As expected, misfolded MalS was found in the pellet fraction of shocked cells. In dsbA degP double null mutants, only reduced MalS was detected. This was in agreement with the previous finding that nearly 60% of MalS activity was detected in dsbA mutants in comparison to wild-type cells after growth at 28☌ ( Table 1). In dsbA mutant background, most of the oxidized form of MalS was present in the supernatant, and smaller amounts of oxidized and reduced MalS were found in the pellet fraction. In agreement with this fact, properly folded MalS was released by cold osmotic shock from wild-type cells. ![]()
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