title: Dimerization of the Pseudomonas aeruginosa translocator chaperone PcrH is required for stability, not function.
abstract: Type III secretion systems rely on hydrophobic translocator proteins that form a pore in the host cell membrane to deliver effector proteins into targeted host cells. These translocator proteins are stabilized in the cytoplasm and targeted for export with the help of specific chaperone proteins. In Pseudomonas aeruginosa, the chaperone of the pore-forming translocator proteins is PcrH. Although all translocator chaperones dimerize, the location of the dimerization interface is in dispute. Moreover, it has been reported that interfering with dimerization interferes with chaperone function. However, binding of P. aeruginosa chaperone PcrH to its cognate secretion substrate, PopD, results in dissociation of the PcrH dimer in vitro, arguing that dimerization of PcrH is likely not important for substrate binding or targeting translocators for export. We demonstrate that PcrH dimerization occurs in vivo in P. aeruginosa and used a genetic screen to identify a dimerization mutant of PcrH. The mutant protein is fully functional in that it can both stabilize PopB and PopD in the cytoplasm and promote their export via the type III secretion system. The location of the mutation suggests that the dimerization interface of PcrH mirrors that of the Yersinia homolog SycD and not the dimerization interface that had previously been reported for PcrH based on crystallographic evidence. Finally, we present data that the dimerization mutant of PcrH is less stable than the wild-type protein in P. aeruginosa, suggesting that the function of dimerization is stabilization of PcrH in the absence of its cognate cargo.
authors: Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.
authors: Amanda G
authors: AG
authors: Tomalka
authors: Stephanie E
authors: SE
authors: Zmina
authors: Charles M
authors: CM
authors: Stopford
authors: Arne
authors: A
authors: Rietsch
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D055550:Protein Stability; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000378:metabolism* / Q000472:pathogenicity; D020798:Two-Hybrid System Techniques; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D018832:Molecular Chaperones
references: Curr Opin Microbiol. 2003 Feb;6(1):7-14
references: 12615213
references: Mol Microbiol. 2012 Dec;86(6):1464-81
references: 23121689
references: Methods Mol Biol. 2004;261:231-46
references: 15064462
references: Nature. 2006 Nov 30;444(7119):567-73
references: 17136086
references: Dev Cell. 2003 Feb;4(2):253-63
references: 12586068
references: Physiology (Bethesda). 2005 Oct;20:326-39
references: 16174872
references: J Biol Chem. 2013 Feb 8;288(6):4048-55
references: 23233673
references: EMBO J. 2003 Oct 1;22(19):4957-67
references: 14517235
references: Nucleic Acids Res. 1990 Aug 11;18(15):4631
references: 2201955
references: J Biol Chem. 2010 Dec 17;285(51):39965-75
references: 20937829
references: J Biol Chem. 2010 Jul 23;285(30):23224-32
references: 20385547
references: J Infect Dis. 2003 Dec 15;188(12):1909-21
references: 14673772
references: J Bacteriol. 2008 Apr;190(8):2726-38
references: 18039770
references: FEMS Microbiol Lett. 2006 Mar;256(1):57-66
references: 16487320
references: Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9661-6
references: 19478065
references: Mol Microbiol. 2008 Jun;68(5):1085-95
references: 18430138
references: BMC Struct Biol. 2010 Jul 15;10:21
references: 20633281
references: Biochemistry. 2007 Jul 10;46(27):8128-37
references: 17571858
references: Mol Microbiol. 2010 Nov;78(4):916-31
references: 20815825
references: Mol Microbiol. 2010 Feb;75(4):924-41
references: 20487288
references: J Mol Biol. 2008 Jan 25;375(4):997-1012
references: 18054956
references: Mol Microbiol. 2006 Jan;59(1):31-44
references: 16359316
references: Proc Natl Acad Sci U S A. 2005 Aug 2;102(31):11082-7
references: 16043713