title: Unraveling sterol-dependent membrane phenotypes by analysis of protein abundance-ratio distributions in different membrane fractions under biochemical and endogenous sterol depletion.
abstract: During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.
authors.affiliation: Max Planck Institute of molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany;
authors.forename: Henrik
authors.initials: H
authors.lastname: Zauber
authors.forename: Witold
authors.initials: W
authors.lastname: Szymanski
authors.forename: Waltraud X
authors.initials: WX
authors.lastname: Schulze
mesh_terms: D000465:Algorithms; D017360:Arabidopsis / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000235:genetics; D002458:Cell Fractionation; D002853:Chromatography, Liquid; D021962:Membrane Microdomains / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008780:Methyltransferases / Q000172:deficiency / Q000235:genetics; D058977:Molecular Sequence Annotation; D009154:Mutation; D059828:Plant Cells / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D018515:Plant Leaves / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D015203:Reproducibility of Results; D013261:Sterols / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000494:pharmacology
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D013261:Sterols; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D008780:Methyltransferases; C474634:SMT1 protein, Arabidopsis
references.citation: Prog Lipid Res. 2012 Jul;51(3):272-99
references.pmid: 22554527
references.citation: Plant J. 2004 Mar;37(6):914-39
references.pmid: 14996223
references.citation: Expert Rev Proteomics. 2011 Apr;8(2):263-77
references.pmid: 21501018
references.citation: Proteomics. 2009 Apr;9(7):1916-24
references.pmid: 19260003
references.citation: Biochim Biophys Acta. 2012 Aug;1821(8):1059-67
references.pmid: 22230596
references.citation: J Biol Chem. 2010 Dec 10;285(50):39140-9
references.pmid: 20843791
references.citation: Mol Cell Proteomics. 2006 Aug;5(8):1396-411
references.pmid: 16648627
references.citation: Plant Cell. 2002 Sep;14(9):1995-2000
references.pmid: 12215500
references.citation: J Proteome Res. 2012 Nov 2;11(11):5548-55
references.pmid: 22978295
references.citation: J Biol Chem. 1998 Aug 28;273(35):22298-304
references.pmid: 9712847
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11411-6
references.pmid: 21709267
references.citation: Traffic. 2005 Sep;6(9):725-40
references.pmid: 16101677
references.citation: J Invest Dermatol. 2011 Jan;131(1):46-58
references.pmid: 20882041
references.citation: Plant Physiol. 2005 Jul;138(3):1195-204
references.pmid: 16009995
references.citation: Mol Cell Proteomics. 2004 Jun;3(6):608-14
references.pmid: 15034119
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2343-8
references.pmid: 20133878
references.citation: Proteomics. 2009 Jan;9(1):138-47
references.pmid: 19053141
references.citation: Genome Biol. 2004;5(6):R41
references.pmid: 15186492
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13964-9
references.pmid: 14612561
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Annu Rev Nutr. 2009;29:257-82
references.pmid: 19400697
references.citation: Biol Chem. 2003 Sep;384(9):1259-63
references.pmid: 14515986
references.citation: Nat Biotechnol. 2008 Dec;26(12):1367-72
references.pmid: 19029910
references.citation: Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16645-50
references.pmid: 19805351
references.citation: Annu Rev Biophys Biomol Struct. 2003;32:257-83
references.pmid: 12543707
references.citation: J Chem Phys. 2010 Apr 7;132(13):135104
references.pmid: 20387961
references.citation: Plant Cell Physiol. 2009 Feb;50(2):341-59
references.pmid: 19106119
references.citation: Plant Physiol. 2005 Jan;137(1):104-16
references.pmid: 15618420
references.citation: Sci STKE. 2003 Jun 17;2003(187):RE9
references.pmid: 12815191
references.citation: J Biol Chem. 2003 Sep 5;278(36):34505-16
references.pmid: 12807886
references.citation: Planta. 2005 Dec;223(1):5-19
references.pmid: 16136329
references.citation: Genes Dev. 2000 Jun 15;14(12):1485-97
references.pmid: 10859167
references.citation: Proc Natl Acad Sci U S A. 2013 May 14;110(20):8296-301
references.pmid: 23630285
references.citation: Mol Plant Microbe Interact. 2011 Jan;24(1):7-12
references.pmid: 21138374
references.citation: Mol Cell Proteomics. 2009 Apr;8(4):612-23
references.pmid: 19036721
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10005-10
references.pmid: 18621689
references.citation: Annu Rev Biophys. 2010;39:207-26
references.pmid: 20192775
references.citation: J Biol Chem. 2004 Aug 27;279(35):36277-86
references.pmid: 15190066
references.citation: Anal Chem. 1998 Dec 15;70(24):5150-8
references.pmid: 9868912
references.citation: Cell Mol Life Sci. 2005 Oct;62(19-20):2228-40
references.pmid: 16091845
references.citation: FASEB J. 2008 Nov;22(11):3980-91
references.pmid: 18676403
references.citation: J Exp Bot. 2007;58(10):2503-12
references.pmid: 17526915
references.citation: Plant Cell. 2009 May;21(5):1541-55
references.pmid: 19470590
references.citation: Proteomics. 2006 Oct;6(20):5409-17
references.pmid: 16972286
references.citation: Plant Cell Physiol. 2004 Nov;45(11):1543-56
references.pmid: 15574830
references.citation: Plant Physiol. 2010 Jun;153(2):741-56
references.pmid: 20421456
references.citation: Database (Oxford). 2011 Mar 29;2011:bar009
references.pmid: 21447597
references.citation: Nat Rev Mol Cell Biol. 2000 Oct;1(1):31-9
references.pmid: 11413487
references.citation: Methods Mol Biol. 2007;406:179-212
references.pmid: 18287693
references.citation: Trends Plant Sci. 2003 Jun;8(6):250-1
references.pmid: 12818657
references.citation: Annu Rev Cell Dev Biol. 2004;20:481-504
references.pmid: 15473849
references.citation: Plant Physiol. 2003 Jun;132(2):568-77
references.pmid: 12805588
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D1185-91
references.pmid: 23180787
references.citation: Nat Protoc. 2007;2(9):2159-65
references.pmid: 17853872
references.citation: Biochim Biophys Acta. 2007 Jun;1768(6):1311-24
references.pmid: 17493580
references.citation: Plant Cell. 2011 Apr;23(4):1191-3
references.pmid: 21531862
references.citation: Trends Cell Biol. 2007 Aug;17(8):394-402
references.pmid: 17766116
references.citation: Plant J. 2004 Apr;38(2):227-43
references.pmid: 15078327
references.citation: Plant J. 2002 Jul;31(1):61-73
references.pmid: 12100483
references.citation: Nat Methods. 2013 Mar;10(3):259-64
references.pmid: 23396283
references.citation: FEBS Lett. 2010 May 3;584(9):1685-93
references.pmid: 20036659
references.citation: Annu Rev Plant Physiol Plant Mol Biol. 1998 Jun;49:427-451
references.pmid: 15012241
references.citation: EMBO J. 2001 Aug 15;20(16):4332-6
references.pmid: 11500359
references.citation: Science. 2012 Aug 31;337(6098):1047-9
references.pmid: 22936764
references.citation: Plant Cell. 2012 May;24(5):2105-22
references.pmid: 22589463
references.citation: Langmuir. 2012 Nov 27;28(47):16327-37
references.pmid: 23121205
references.citation: Plant Physiol. 2002 Jun;129(2):486-99
references.pmid: 12068095
references.citation: Anal Chem. 2003 Feb 1;75(3):663-70
references.pmid: 12585499
references.citation: Biochem J. 1998 Oct 15;335 ( Pt 2):433-40
references.pmid: 9761744