title: Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome with syringolin A.
abstract: As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra.
authors.affiliation: From the ‡Department of Biology, Universitaetstrasse 2, ETH Zurich, CH-8092 Zurich, Switzerland;
authors.forename: Julia
authors.initials: J
authors.lastname: Svozil
authors.affiliation: From the ‡Department of Biology, Universitaetstrasse 2, ETH Zurich, CH-8092 Zurich, Switzerland;
authors.forename: Matthias
authors.initials: M
authors.lastname: Hirsch-Hoffmann
authors.affiliation: §Institute of Plant Biology, Zollikerstrasse 107, University of Zurich, CH-8008 Zurich, Switzerland.
authors.forename: Robert
authors.initials: R
authors.lastname: Dudler
authors.affiliation: From the ‡Department of Biology, Universitaetstrasse 2, ETH Zurich, CH-8092 Zurich, Switzerland;
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Gruissem
authors.affiliation: From the ‡Department of Biology, Universitaetstrasse 2, ETH Zurich, CH-8092 Zurich, Switzerland; kbaerenfaller@ethz.ch.
authors.forename: Katja
authors.initials: K
authors.lastname: Baerenfaller
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development; D018506:Gene Expression Regulation, Plant; D006801:Humans; D010456:Peptides, Cyclic / Q000008:administration & dosage; D010940:Plant Proteins / Q000096:biosynthesis*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects*; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D040901:Proteomics; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects*
chemical_list: D010456:Peptides, Cyclic; D010940:Plant Proteins; D025801:Ubiquitin; C407267:syringolin A; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease
references.citation: BMC Plant Biol. 2012 Sep 12;12:164
references.pmid: 22970698
references.citation: Science. 2012 Nov 2;338(6107):622-3
references.pmid: 23118178
references.citation: Plant J. 2010 May 1;62(4):539-48
references.pmid: 20536787
references.citation: Mol Plant Microbe Interact. 2012 Sep;25(9):1198-208
references.pmid: 22852810
references.citation: Plant Physiol. 2008 Aug;147(4):1560-4
references.pmid: 18678747
references.citation: Proc Natl Acad Sci U S A. 2010 May 25;107(21):9909-14
references.pmid: 20457921
references.citation: Mol Cell Proteomics. 2013 Mar;12(3):825-31
references.pmid: 23266961
references.citation: Plant Cell. 2008 Jul;20(7):1899-914
references.pmid: 18664614
references.citation: Nat Methods. 2007 Mar;4(3):207-14
references.pmid: 17327847
references.citation: Ann Bot. 2007 May;99(5):787-822
references.pmid: 17220175
references.citation: Nat Rev Mol Cell Biol. 2001 Mar;2(3):195-201
references.pmid: 11265249
references.citation: Plant Cell. 2008 Jun;20(6):1693-707
references.pmid: 18552202
references.citation: J Proteome Res. 2009 Sep;8(9):4243-51
references.pmid: 19603825
references.citation: Plant Physiol. 2013 Jun;162(2):897-906
references.pmid: 23645632
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D1063-9
references.pmid: 23203882
references.citation: Cell. 2010 Nov 24;143(5):677-81
references.pmid: 21111228
references.citation: Front Plant Sci. 2012 Jun 11;3:123
references.pmid: 22701464
references.citation: Plant Cell. 2011 Feb;23(2):471-85
references.pmid: 21343415
references.citation: Nature. 2006 Mar 23;440(7083):551-5
references.pmid: 16554822
references.citation: Science. 2008 May 16;320(5878):938-41
references.pmid: 18436743
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13238-43
references.pmid: 16916932
references.citation: Nat Protoc. 2007;2(4):953-71
references.pmid: 17446895
references.citation: Nat Biotechnol. 2010 Aug;28(8):868-73
references.pmid: 20639865
references.citation: Plant Cell. 2005 Oct;17(10):2723-37
references.pmid: 16169895
references.citation: Biostatistics. 2003 Apr;4(2):249-64
references.pmid: 12925520
references.citation: Plant J. 2010 Apr 1;62(1):160-70
references.pmid: 20042019
references.citation: J Proteome Res. 2009 Jan;8(1):104-12
references.pmid: 18954100
references.citation: Mol Syst Biol. 2012;8:606
references.pmid: 22929616
references.citation: Science. 2011 Jun 17;332(6036):1439-42
references.pmid: 21680842
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D109-14
references.pmid: 22080510
references.citation: Planta. 2006 Mar;223(4):684-97
references.pmid: 16200408
references.citation: Plant J. 2010 Mar;61(6):1029-40
references.pmid: 20409276
references.citation: Mol Syst Biol. 2009;5:314
references.pmid: 19888209
references.citation: Annu Rev Biochem. 2001;70:503-33
references.pmid: 11395416
references.citation: FEBS Lett. 2009 Dec 17;583(24):3966-73
references.pmid: 19850042
references.citation: Nat Rev Mol Cell Biol. 2008 Dec;9(12):944-57
references.pmid: 19002207
references.citation: Mol Syst Biol. 2010 Aug 24;6:400
references.pmid: 20739923
references.citation: PLoS Genet. 2013;9(1):e1003196
references.pmid: 23341779
references.citation: Plant Physiol. 2003 Jun;132(2):861-9
references.pmid: 12805616
references.citation: Science. 2012 Nov 2;338(6107):655-9
references.pmid: 23118188
references.citation: Nat Biotechnol. 2003 Aug;21(8):921-6
references.pmid: 12872131
references.citation: Integr Biol (Camb). 2011 Mar;3(3):225-37
references.pmid: 21264403
references.citation: Mol Cell Proteomics. 2011 Oct;10(10):M111.013284
references.pmid: 21890473
references.citation: Plant Cell. 2009 Dec;21(12):3984-4001
references.pmid: 20028838
references.citation: Plant Physiol. 2009 Jan;149(1):561-74
references.pmid: 18987211
references.citation: Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12955-60
references.pmid: 21768351
references.citation: Dev Biol. 1999 Nov 15;215(2):407-19
references.pmid: 10545247
references.citation: Plant Cell. 2011 Nov;23(11):3911-28
references.pmid: 22128122
references.citation: Mol Biosyst. 2009 Dec;5(12):1512-26
references.pmid: 20023718
references.citation: Mol Plant Microbe Interact. 2005 Sep;18(9):923-37
references.pmid: 16167763
references.citation: Plant Mol Biol. 2008 Sep;68(1-2):145-58
references.pmid: 18535787
references.citation: Plant Mol Biol. 2006 Nov;62(4-5):561-78
references.pmid: 16941219
references.citation: Mol Cells. 2011 Mar;31(3):201-8
references.pmid: 21347703
references.citation: Trends Plant Sci. 2012 Jun;17(6):332-40
references.pmid: 22401845
references.citation: Curr Opin Plant Biol. 2010 Aug;13(4):402-8
references.pmid: 20471305
references.citation: Plant Cell. 2008 Jan;20(1):213-27
references.pmid: 18178771
references.citation: Plant J. 2007 Jan;49(2):228-37
references.pmid: 17241447
references.citation: J Exp Bot. 2009;60(11):3067-73
references.pmid: 19429840
references.citation: Trends Biochem Sci. 2003 Nov;28(11):598-603
references.pmid: 14607090
references.citation: J Exp Bot. 2009;60(4):1123-32
references.pmid: 19276192
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D1185-91
references.pmid: 23180787
references.citation: Plant J. 1999 Nov;20(4):503-8
references.pmid: 10607302
references.citation: Plant Cell. 2001 Jan;13(1):153-61
references.pmid: 11158536
references.citation: Mol Cell Proteomics. 2007 Apr;6(4):601-10
references.pmid: 17272265
references.citation: Mol Syst Biol. 2011 Jul 19;7:514
references.pmid: 21772262
references.citation: Bioinformatics. 2008 Jul 15;24(14):1650-1
references.pmid: 18511468
references.citation: Plant Cell. 2013 May;25(5):1523-40
references.pmid: 23667124
references.citation: Cell. 2006 Oct 6;127(1):99-111
references.pmid: 17018280
references.citation: Reproduction. 2005 Mar;129(3):269-82
references.pmid: 15749954
references.citation: Plant Physiol. 2007 Jan;143(1):199-212
references.pmid: 17098851
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D1202-10
references.pmid: 22140109
references.citation: Biochem Soc Trans. 2009 Oct;37(Pt 5):937-53
references.pmid: 19754430
references.citation: Nature. 2011 May 19;473(7347):337-42
references.pmid: 21593866
references.citation: Nat Rev Mol Cell Biol. 2009 Jun;10(6):385-97
references.pmid: 19424292
references.citation: Plant J. 2007 Oct;52(2):309-21
references.pmid: 17714429
references.citation: Plant J. 2009 Jul;59(2):344-58
references.pmid: 19292762
references.citation: Autophagy. 2011 Mar;7(3):279-96
references.pmid: 21189453
references.citation: Trends Plant Sci. 2012 Sep;17(9):526-37
references.pmid: 22694835
references.citation: Mol Cell Proteomics. 2012 Mar;11(3):M111.014035
references.pmid: 22311637
references.citation: Nature. 2008 Apr 10;452(7188):755-8
references.pmid: 18401409
references.citation: Curr Opin Chem Biol. 2008 Aug;12(4):434-40
references.pmid: 18656549
references.citation: J Biol Chem. 2008 Jun 6;283(23):15869-77
references.pmid: 18420588
references.citation: Annu Rev Plant Biol. 2011;62:299-334
references.pmid: 21370976