title: Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy.
abstract: Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection-Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection-Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.
authors.affiliation: Department of Biophysics, Ruhr-University Bochum, 44801 Bochum, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Gavriljuk
authors.forename: Aymelt
authors.initials: A
authors.lastname: Itzen
authors.forename: Roger S
authors.initials: RS
authors.lastname: Goody
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
mesh_terms: D000818:Animals; D002417:Cattle; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D007700:Kinetics; D016952:Legionella pneumophila / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism*; D010767:Phosphorylcholine / Q000378:metabolism; D054876:Prenylation; D012441:Saccharomyces cerevisiae; D013055:Spectrophotometry, Infrared / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D022161:Transport Vesicles / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D010767:Phosphorylcholine; D020691:rab GTP-Binding Proteins
keywords: Rab recycling; geranylgeranyl; lipid bilayers; vesicle spreading
references.citation: Science. 2003 Oct 24;302(5645):646-50
references.pmid: 14576435
references.citation: Nature. 1996 May 2;381(6577):42-8
references.pmid: 8609986
references.citation: Curr Biol. 2006 Sep 5;16(17):1719-25
references.pmid: 16950109
references.citation: Biochemistry. 1994 Mar 15;33(10):3014-22
references.pmid: 8130214
references.citation: Nat Rev Mol Cell Biol. 2005 Mar;6(3):209-20
references.pmid: 15738987
references.citation: EMBO J. 2006 Jan 11;25(1):13-23
references.pmid: 16395334
references.citation: Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21348-53
references.pmid: 23236136
references.citation: Q Rev Biophys. 1997 Nov;30(4):365-429
references.pmid: 9634652
references.citation: EMBO J. 2012 Apr 4;31(7):1774-84
references.pmid: 22307087
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12294-9
references.pmid: 17640890
references.citation: Nat Rev Mol Cell Biol. 2009 Aug;10(8):513-25
references.pmid: 19603039
references.citation: Nat Chem Biol. 2010 Jul;6(7):534-40
references.pmid: 20512138
references.citation: FEBS J. 2008 Dec;275(23):5910-8
references.pmid: 19021766
references.citation: J Biol Chem. 2009 May 8;284(19):13185-92
references.pmid: 19240028
references.citation: Science. 2010 Aug 20;329(5994):946-9
references.pmid: 20651120
references.citation: Nat Genet. 1998 Jun;19(2):134-9
references.pmid: 9620768
references.citation: J Biol Chem. 2008 Jun 27;283(26):18377-84
references.pmid: 18426803
references.citation: Biophys J. 2006 Aug 15;91(4):1380-7
references.pmid: 16731563
references.citation: Langmuir. 2006 Apr 11;22(8):3497-505
references.pmid: 16584220
references.citation: Nature. 2011 Aug 07;477(7362):103-6
references.pmid: 21822290
references.citation: Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5621-6
references.pmid: 22411835
references.citation: Biochemistry. 1995 Mar 21;34(11):3813-22
references.pmid: 7893678
references.citation: Traffic. 2001 Aug;2(8):532-43
references.pmid: 11489211
references.citation: Angew Chem Int Ed Engl. 2010 Jul 26;49(32):5416-24
references.pmid: 20818765
references.citation: Science. 2008 Jun 20;320(5883):1651-4
references.pmid: 18566289
references.citation: J Am Chem Soc. 2013 Mar 13;135(10):4079-87
references.pmid: 23414555
references.citation: J Biol Chem. 1996 Mar 8;271(10):5289-92
references.pmid: 8621375
references.citation: J Biol Chem. 1997 Sep 12;272(37):23138-43
references.pmid: 9287316
references.citation: J Cell Biol. 2013 Feb 4;200(3):287-300
references.pmid: 23382462
references.citation: Mol Biol Cell. 2006 Aug;17(8):3494-507
references.pmid: 16687576
references.citation: Biophys J. 1991 Jun;59(6):1261-72
references.pmid: 1873463
references.citation: Biochim Biophys Acta. 1999 Jul 6;1422(2):105-85
references.pmid: 10393271
references.citation: Biophys J. 2012 Oct 3;103(7):1585-93
references.pmid: 23062351