title: Sequential steps in the assembly of the multimeric outer membrane secretin PulD.
abstract: Investigations into protein folding are largely dominated by studies on monomeric proteins. However, the transmembrane domain of an important group of membrane proteins is only formed upon multimerization. Here, we use in vitro translation-coupled folding and insertion into artificial liposomes to investigate kinetic steps in the assembly of one such protein, the outer membrane secretin PulD of the bacterial type II secretion system. Analysis of the folding kinetics, measured by the acquisition of distinct determinants of the native state, provides unprecedented evidence for a sequential multistep process initiated by membrane-driven oligomerization. The effects of varying the lipid composition of the liposomes indicate that PulD first forms a "prepore" structure that attains the native state via a conformational switch.
authors.affiliation: From the Molecular Genetics Unit, Departments of Microbiology and Structural Biology and Chemistry, and CNRS ERL3526, Institut Pasteur, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Gerard H M
authors.initials: GHM
authors.lastname: Huysmans
authors.affiliation: From the Molecular Genetics Unit, Departments of Microbiology and Structural Biology and Chemistry, and CNRS ERL3526, Institut Pasteur, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Guilvout
authors.affiliation: From the Molecular Genetics Unit, Departments of Microbiology and Structural Biology and Chemistry, and CNRS ERL3526, Institut Pasteur, rue du Dr. Roux, 75724 Paris Cedex 15, France. Electronic address: max@pasteur.fr.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D058947:Bacterial Secretion Systems / Q000502:physiology*; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry; D017510:Protein Folding; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary
chemical_list: D001425:Bacterial Outer Membrane Proteins; D058947:Bacterial Secretion Systems; D008081:Liposomes; C061377:PulD protein, Klebsiella pneumoniae
keywords: Kinetics; Membrane Proteins; Phospholipid Vesicle; Protein Self-assembly; Protein Synthesis; Secretin; Type II Secretion
references.citation: J Biol Chem. 2013 Jan 11;288(2):1214-25
references.pmid: 23188826
references.citation: Biochemistry. 1992 Apr 21;31(15):3738-50
references.pmid: 1567828
references.citation: Mol Microbiol. 2007 Jun;64(5):1350-7
references.pmid: 17542925
references.citation: J Bacteriol. 2012 Sep;194(18):4951-8
references.pmid: 22773793
references.citation: Microbiol Mol Biol Rev. 2003 Dec;67(4):593-656
references.pmid: 14665678
references.citation: Curr Opin Struct Biol. 2012 Jun;22(3):342-9
references.pmid: 22658510
references.citation: Curr Opin Struct Biol. 2006 Apr;16(2):230-6
references.pmid: 16563740
references.citation: Science. 2007 Sep 14;317(5844):1548-51
references.pmid: 17717151
references.citation: J Biol Chem. 2005 Nov 11;280(45):37732-41
references.pmid: 16129681
references.citation: J Mol Biol. 2008 Sep 26;382(1):13-23
references.pmid: 18616949
references.citation: Biochemistry. 1995 Feb 14;34(6):1921-9
references.pmid: 7849052
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4099-104
references.pmid: 20133664
references.citation: Biol Chem. 2012 Nov;393(11):1215-30
references.pmid: 23096346
references.citation: PLoS Pathog. 2012;8(7):e1002787
references.pmid: 22792065
references.citation: Mol Microbiol. 2011 May;80(3):655-65
references.pmid: 21338419
references.citation: Macromol Biosci. 2010 Jul 7;10(7):763-7
references.pmid: 20491126
references.citation: Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):19802-7
references.pmid: 21041662
references.citation: J Biol Chem. 1987 Oct 5;262(28):13742-9
references.pmid: 3308882
references.citation: J Mol Biol. 2006 Apr 21;358(1):120-31
references.pmid: 16497324
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4065-70
references.pmid: 14990786
references.citation: J Mol Biol. 2012 Feb 24;416(3):453-64
references.pmid: 22245579
references.citation: J Am Chem Soc. 2011 Jul 27;133(29):11389-98
references.pmid: 21682279
references.citation: Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14133-8
references.pmid: 21831834
references.citation: J Biol Chem. 2008 Sep 26;283(39):26748-58
references.pmid: 18641391
references.citation: Biochemistry. 2009 Nov 3;48(43):10235-45
references.pmid: 19780589
references.citation: Microbiology (Reading). 2009 Jun;155(Pt 6):1847-1857
references.pmid: 19383696
references.citation: Nat Rev Microbiol. 2012 Jul 02;10(8):563-74
references.pmid: 22751485
references.citation: Adv Exp Med Biol. 2012;747:122-36
references.pmid: 22949115
references.citation: Biophys J. 2011 May 4;100(9):2131-40
references.pmid: 21539780
references.citation: N Biotechnol. 2011 Apr 30;28(3):262-71
references.pmid: 20637904
references.citation: J Biol Chem. 1992 Jul 5;267(19):13391-7
references.pmid: 1618841
references.citation: EMBO J. 2000 May 15;19(10):2229-36
references.pmid: 10811614
references.citation: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10174-7
references.pmid: 21606332
references.citation: Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):18970-5
references.pmid: 18025476
references.citation: Biophys Chem. 2002 Dec 10;101-102:113-31
references.pmid: 12487994
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):11890-5
references.pmid: 21730166
references.citation: EMBO J. 1996 Mar 1;15(5):978-88
references.pmid: 8605893
references.citation: Nature. 2010 Nov 18;468(7322):447-51
references.pmid: 21037563
references.citation: J Bacteriol. 1999 Dec;181(23):7212-20
references.pmid: 10572123
references.citation: Res Microbiol. 2011 Feb-Mar;162(2):180-90
references.pmid: 21256212
references.citation: Biochemistry. 2010 Jan 19;49(2):279-86
references.pmid: 20000688
references.citation: Mol Microbiol. 2005 Jul;57(2):326-46
references.pmid: 15978068
references.citation: Mol Microbiol. 1997 May;24(3):465-75
references.pmid: 9179841
references.citation: J Mol Biol. 2013 Sep 9;425(17):3178-91
references.pmid: 23796519
references.citation: J Bacteriol. 2012 Apr;194(7):1646-58
references.pmid: 22247509
references.citation: J Biol Chem. 2012 Jun 1;287(23):19082-93
references.pmid: 22523076
references.citation: Biochim Biophys Acta. 2012 Apr;1818(4):942-50
references.pmid: 22178866
references.citation: Biochemistry. 1996 Oct 8;35(40):12993-3000
references.pmid: 8855933
references.citation: Mol Microbiol. 1998 Jan;27(1):209-19
references.pmid: 9466268
references.citation: Biochemistry. 1981 Apr 28;20(9):2417-24
references.pmid: 7236611
references.citation: Annu Rev Biochem. 2011;80:189-210
references.pmid: 21370981
references.citation: Res Microbiol. 2013 Jun;164(5):390-6
references.pmid: 23567323
references.citation: EMBO J. 2006 Nov 15;25(22):5241-9
references.pmid: 17082772
references.citation: Cell Mol Life Sci. 2010 Jun;67(11):1779-98
references.pmid: 20101433
references.citation: J Mol Neurosci. 2004;23(1-2):41-8
references.pmid: 15126691
references.citation: Mol Membr Biol. 2009 Apr;26(3):136-45
references.pmid: 19225986
references.citation: Curr Opin Struct Biol. 2012 Apr;22(2):208-16
references.pmid: 22425326
references.citation: Proc Natl Acad Sci U S A. 2012 May 22;109(21):8121-6
references.pmid: 22566663
references.citation: Curr Opin Struct Biol. 2011 Feb;21(1):42-9
references.pmid: 21075614
references.citation: J Biol Chem. 2011 Nov 11;286(45):38833-43
references.pmid: 21878629