title: Distinct regulatory mechanisms balance DegP proteolysis to maintain cellular fitness during heat stress.
abstract: Intracellular proteases combat proteotoxic stress by degrading damaged proteins, but their activity must be carefully controlled to maintain cellular fitness. The activity of Escherichia coli DegP, a highly conserved periplasmic protease, is regulated by substrate-dependent allosteric transformations between inactive and active trimer conformations and by the formation of polyhedral cages that confine the active sites within a proteolytic chamber. Here, we investigate how these distinct control mechanisms contribute to bacterial fitness under heat stress. We found that mutations that increase or decrease the equilibrium population of active DegP trimers reduce high-temperature fitness, that a mutation that blocks cage formation causes a mild fitness decrease, and that combining mutations that stabilize active DegP and block cage formation generates a lethal rogue protease. This lethality is suppressed by an extragenic mutation that prevents covalent attachment of an abundant outer-membrane lipoprotein to peptidoglycan and makes this protein an inhibitor of the rogue protease. Lethality is also suppressed by intragenic mutations that stabilize inactive DegP trimers. In combination, our results suggest that allosteric control of active and inactive conformations is the primary mechanism that regulates DegP proteolysis and fitness, with cage formation providing an additional layer of cellular protection against excessive protease activity.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Seokhee
authors.initials: S
authors.lastname: Kim
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D050296:Microbial Viability / Q000235:genetics; D009154:Mutation; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics / Q000502:physiology*
chemical_list: D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; C061682:DegP protease; D012697:Serine Endopeptidases
keywords: DegP; HtrA; Lpp; cellular fitness; protein quality control; proteolysis
references.citation: Nature. 2002 Mar 28;416(6879):455-9
references.pmid: 11919638
references.citation: Curr Protoc Mol Biol. 2007 Jul;Chapter 1:Unit 1.17
references.pmid: 18265391
references.citation: J Bacteriol. 2012 May;194(9):2142-51
references.pmid: 22287519
references.citation: Annu Rev Biochem. 2011;80:587-612
references.pmid: 21469952
references.citation: Science. 2006 Mar 17;311(5767):1615-7
references.pmid: 16543462
references.citation: Annu Rev Biochem. 1978;47:481-532
references.pmid: 354502
references.citation: EMBO J. 1997 Dec 1;16(23):6947-55
references.pmid: 9384574
references.citation: ACS Chem Biol. 2011 Nov 18;6(11):1205-13
references.pmid: 21866931
references.citation: FEBS Lett. 1998 Nov 13;439(1-2):51-4
references.pmid: 9849875
references.citation: J Bacteriol. 1997 May;179(9):2857-62
references.pmid: 9139900
references.citation: Eur J Biochem. 1969 Oct;10(3):426-38
references.pmid: 4899922
references.citation: J Bacteriol. 1989 Mar;171(3):1574-84
references.pmid: 2537822
references.citation: J Biol Chem. 2010 Oct 29;285(44):34039-47
references.pmid: 20739286
references.citation: J Biol Chem. 1972 Dec 25;247(24):8154-9
references.pmid: 4565677
references.citation: Mol Microbiol. 2009 Apr;72(2):368-79
references.pmid: 19298369
references.citation: Proc Natl Acad Sci U S A. 2012 May 8;109(19):7263-8
references.pmid: 22529381
references.citation: Nat Rev Mol Cell Biol. 2011 Mar;12(3):152-62
references.pmid: 21326199
references.citation: J Mol Biol. 1978 Jun 5;121(4):493-506
references.pmid: 353292
references.citation: J Bacteriol. 1981 Jan;145(1):654-6
references.pmid: 7007330
references.citation: Biochim Biophys Acta. 1975 Oct 31;415(3):335-77
references.pmid: 52377
references.citation: Nat Med. 2005 Oct;11(10):1082-7
references.pmid: 16200071
references.citation: J Biol Chem. 2005 Jan 14;280(2):974-83
references.pmid: 15513925
references.citation: J Bacteriol. 1989 May;171(5):2689-96
references.pmid: 2540154
references.citation: J Bacteriol. 2000 Jan;182(2):371-6
references.pmid: 10629182
references.citation: Cell. 2011 Apr 1;145(1):67-78
references.pmid: 21458668
references.citation: Mol Cell. 2009 Jan 16;33(1):64-74
references.pmid: 19150428
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11939-44
references.pmid: 18697939
references.citation: Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7702-7
references.pmid: 18505836
references.citation: Nature. 2008 Jun 12;453(7197):885-90
references.pmid: 18496527
references.citation: Mol Microbiol. 1997 Oct;26(2):209-21
references.pmid: 9383148
references.citation: Biochemistry. 2008 Jun 10;47(23):6092-102
references.pmid: 18479146
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: Nature. 2013 Nov 21;503(7476):365-70
references.pmid: 24226776