Overexpression of Protease-Deficient DegP _S210A Rescues the Lethal Phenotype of Escherichia coli OmpF Assembly Mutants in a degP Background

Abstract

Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP S210A protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP S210A was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP S210A also fractionated. These results suggested that the DegP S210A -mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.

The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures

“…The complexity of the folding process might be the reason for the lack of effect of HtrA chaperone activity on AP maturation. A similar observation was presented in the works of Misra et al (2000) and CastilloKeller & Misra (2003). These authors found that overproduced, mutated subunits of outer-membrane porins OmpF or OmpC were sequestered by HtrAS210A but their assembly into active oligomers was not improved.…”

Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

“…Skorko-Glonek et al (2007) have shown that at high temperatures HtrA S210A forms complexes with denatured proteins but does not refold them; rather it prevents the formation of denatured protein aggregates in the periplasm (SkorkoGlonek et al, 2007). HtrA S210A could rescue an E. coli htrA mutant from the lethal effects of the misfolding of the porins OmpF and OmpC (CastilloKeller & Misra, 2003;Misra et al, 2000). This was not accomplished by HtrA S210A correctly folding OmpF or OmpC, suggesting that HtrA S210A was sequestering the misfolded proteins.…”

Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival

“…Third, DegP cages may facilitate additional biological activities. For example, degP S210A supports growth at 42°C better than a ΔdegP allele, a property that has been proposed to result from chaperone activity (13) or sequestration of toxic misfolded proteins (26,27). Finally, DegP cages may allow more efficient degradation of large protein substrates, even though this activity is not required for high-temperature survival.…”