Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background.
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.
Department of Microbiology, Arizona State University, Tempe, Arizona 85287, USA. rajeev.misra@asu.edu
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D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015870:Gene Expression; D006360:Heat-Shock Proteins; D007700:Kinetics; D016296:Mutagenesis; D033903:Periplasmic Proteins; D010641:Phenotype; D010649:Phenylalanine / Q000235:genetics; D018272:Porins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C109237:OmpF protein; D033903:Periplasmic Proteins; D018272:Porins; D010649:Phenylalanine; C061682:DegP protease; D012697:Serine Endopeptidases