Skip to main content
SpringerLink
Log in

Purification of SUMO Conjugates from Arabidopsis for Mass Spectrometry Analysis

  • Protocol
  • First Online:
SUMO

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1475))

Abstract

The posttranslational modification of proteins with small ubiquitin-related modifier (SUMO) is a rapid, robust, and reversible mechanism that impacts a host of eukaryotic processes important to both normal cellular functions and survival during various abiotic and biotic challenges. Essential to defining the breadth of events impacted by SUMOylation is the development of full catalogues of protein targets. Here, we describe a stringent affinity method to purify native SUMO conjugates from the model plant Arabidopsis thaliana based on the expression of modified SUMOs bearing epitope tags. When combined with standard and quantitative mass spectrometric methods, deep datasets of SUMOylated proteins can be acquired. Functional analysis of these lists links SUMO to numerous regulatory events, with an emphasis on those associated with transcription, DNA replication and repair, and chromatin assembly/accessibility.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kurepa J, Walker JM, Smalle J et al (2003) The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress. J Biol Chem 278:6862–6872

    Google Scholar 

  2. Saracco SA, Miller MJ, Kurepa J et al (2007) Genetic analysis of SUMOylation in Arabidopsis: conjugation of SUMO1 and SUMO2 to nuclear proteins is essential. Plant Physiol 145:119–134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Takahashi Y, Iwase M, Konishi M et al (1999) Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast. Biochem Biophys Res Commun 259:582–587

    Article  CAS  PubMed  Google Scholar 

  4. Nacerddine K, Lehembre F, Bhaumik M et al (2005) The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice. Dev Cell 9:769–779

    Article  CAS  PubMed  Google Scholar 

  5. Wang L, Wansleeben C, Zhao S et al (2014) SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development. EMBO Rep 15:878–885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Miura K, Lee J, Miura T et al (2010) SIZ1 controls cell growth and plant development in Arabidopsis through salicylic acid. Plant Cell Physiol 51:103–113

    Article  CAS  PubMed  Google Scholar 

  7. Miura K, Rus A, Sharkhuu A et al (2005) The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proc Natl Acad Sci U S A 102:7760–7765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Miura K, Jin JB, Lee J et al (2007) SIZ1-mediated SUMOylation of ICE1 controls CBF3/DREB1A expression and freezing tolerance in Arabidopsis. Plant Cell 19:1403–1414

    Google Scholar 

  9. Lee J, Nam J, Park HC et al (2007) Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase. Plant J 49:79–90

    Article  CAS  PubMed  Google Scholar 

  10. Jin JB, Hasegawa PM (2008) Flowering time regulation by the SUMO E3 ligase SIZ1. Plant Signal Behav 3:891–892

    Article  PubMed  PubMed Central  Google Scholar 

  11. Miura K, Lee J, Jin JB et al (2009) SUMOylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling. Proc Natl Acad Sci U S A 106:5418–5423

    Google Scholar 

  12. Catala R, Ouyang J, Abreu IA et al (2007) The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth and drought responses. Plant Cell 19:2952–2966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yoo CY, Miura K, Jin JB et al (2006) SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal thermotolerance in Arabidopsis independent of salicylic acid. Plant Physiol 142:1548–1558

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Miura K, Lee J, Gong Q et al (2011) SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation. Plant Physiol 155:1000–1012

    Article  CAS  PubMed  Google Scholar 

  15. Chen C-C, Chen Y-Y, Tang I-C et al (2011) Arabidopsis SUMO E3 ligase SIZ1 is involved in excess copper tolerance. Plant Physiol 156:2225–2234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cheong MS, Park HC, Hong MJ et al (2009) Specific domain structures control abscisic acid-, salicylic acid-, and stress-mediated SIZ1 phenotypes. Plant Physiol 151:1930–1942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jin JB, Jin YH, Lee J et al (2008) The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure. Plant J 53:530–540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tomanov K, Zeschmann A, Hermkes R et al (2014) Arabidopsis PIAL1 and 2 promote SUMO chain formation as E4-type SUMO ligases and are involved in stress responses and sulfur metabolism. Plant Cell 26:4547–4560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ishida T, Fujiwara S, Miura K et al (2009) SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis. Plant Cell 21:2284–2297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Huang L, Yang S, Zhang S et al (2009) The Arabidopsis SUMO E3 ligase AtMMS21, a homologue of NSE2/MMS21, regulates cell proliferation in the root. Plant J 60:666–678

    Article  CAS  PubMed  Google Scholar 

  21. Colby T, Matthäi A, Boeckelmann A et al (2006) SUMO-conjugating and SUMO-deconjugating enzymes from Arabidopsis. Plant Physiol 142:318–332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Miller MJ, Barrett-Wilt GA, Hua Z et al (2010) Proteomic analyses identify a diverse array of nuclear processes affected by small ubiquitin-like modifier conjugation in Arabidopsis. Proc Natl Acad Sci U S A 107:16512–16517

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Perry JJP, Tainer JA, Boddy MN (2008) A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem Sci 33:201–208

    Article  CAS  PubMed  Google Scholar 

  24. Elrouby N, Bonequi MV, Porri A et al (2013) Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions. Proc Natl Acad Sci U S A 110:19956–19961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Murtas G, Reeves PH, Fu Y et al (2003) A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates. Plant Cell 15:2308–2319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Conti L, Price G, O’Donnell E et al (2008) Small ubiquitin-like modifier proteases OVERLY TOLERANT TO SALT1 and -2 regulate salt stress responses in Arabidopsis. Plant Cell 20:2894–2908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Novatchkova M, Tomanov K, Hofmann K et al (2012) Update on SUMOylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison. New Phytol 195:23–31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398:246–251

    Article  CAS  PubMed  Google Scholar 

  29. Novatchkova M, Budhiraja R, Coupland G et al (2004) SUMO conjugation in plants. Planta 220:1–8.

    Google Scholar 

  30. Augustine RC, York SL, Rytz TC, Vierstra RD (2016) Defining the SUMOylation system in maize: SUMOylation is up-regulated during endosperm development and rapidly induced by stress. Plant Physiol doi/10.1104/pp.16.00353

    Google Scholar 

  31. van den Burg HA, Kini RK, Schuurink RC et al (2010) Arabidopsis small ubiquitin-like modifier paralogs have distinct functions in development and defense. Plant Cell 22:1998–2016

    Article  PubMed  PubMed Central  Google Scholar 

  32. Miller MJ, Scalf M, Rytz TC et al (2013) Quantitative proteomics reveals factors regulating RNA biology as dynamic targets of stress-induced SUMOylation in Arabidopsis. Mol Cell Proteomics 12:449–463

    Article  CAS  PubMed  Google Scholar 

  33. Vertegaal ACO, Ogg SC, Jaffray E et al (2004) A proteomic study of SUMO-2 target proteins. J Biol Chem 279:33791–33798

    Article  CAS  PubMed  Google Scholar 

  34. Wohlschlegel JA, Johnson ES, Reed SI et al (2004) Global analysis of protein SUMOylation in Saccharomyces cerevisiae. J Biol Chem 279:45662–45668

    Google Scholar 

  35. Denison C, Rudner AD, Gerber SA et al (2005) A proteomic strategy for gaining insights into protein SUMOylation in yeast. Mol Cell Proteomics 4:246–254

    Google Scholar 

  36. Wohlschlegel JA, Johnson ES, Reed SI et al (2006) Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J Proteome Res 5:761–770

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Vertegaal ACO, Andersen JS, Ogg SC et al (2006) Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol Cell Proteomics 5:2298–2310

    Article  CAS  PubMed  Google Scholar 

  38. Schimmel J, Larsen KM, Matic I et al (2008) The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle. Mol Cell Proteomics 7:2107–2122

    Article  CAS  PubMed  Google Scholar 

  39. Golebiowski F, Matic I, Tatham MH et al (2009) System-wide changes to SUMO modifications in response to heat shock. Sci Signal 2:ra24

    Article  PubMed  Google Scholar 

  40. Blomster HA, Hietakangas V, Wu J et al (2009) Novel proteomics strategy brings insight into the prevalence of SUMO-2 target sites. Mol Cell Proteomics 8:1382–1390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Budhiraja R, Hermkes R, Müller S et al (2009) Substrates related to chromatin and to RNA-dependent processes are modified by Arabidopsis SUMO isoforms that differ in a conserved residue with influence on deSUMOylation. Plant Physiol 149:1529–1540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Matic I, Schimmel J, Hendriks IA et al (2010) Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol Cell 39:641–652

    Article  CAS  PubMed  Google Scholar 

  43. Hendriks IA, D’Souza RC, Chang J-G et al (2015) System-wide identification of wild-type SUMO-2 conjugation sites. Nat Commun 6:7289

    Article  PubMed  PubMed Central  Google Scholar 

  44. Hendriks IA, D’Souza RCJ, Yang B et al (2014) Uncovering global SUMOylation signaling networks in a site-specific manner. Nat Struct Mol Biol 21:927–936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Xiao Z, Chang J-G, Hendriks IA et al (2015) System-wide analysis of SUMOylation dynamics in response to replication stress reveals novel Small Ubiquitin-like Modified target proteins and acceptor lysines relevant for genome stability. Mol Cell Proteomics 14:1419–1434

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hendriks IA, Treffers LW, Verlaan-de Vries M, et al. (2015) SUMO-2 orchestrates chromatin modifiers in response to DNA damage. Cell Rep. 10:1778–1791

    Google Scholar 

  47. Park HC, Choi W, Park HJ et al (2011) Identification and molecular properties of SUMO-binding proteins in Arabidopsis. Mol Cells 32:143–151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. López-Torrejón G, Guerra D, Catalá R et al (2013) Identification of SUMO targets by a novel proteomic approach in plants. J Integr Plant Biol 55:96–107

    Article  PubMed  Google Scholar 

  49. Miller MJ, Vierstra RD (2011) Mass spectrometric identification of SUMO substrates provides insights into heat stress-induced SUMOylation in plants. Plant Signal Behav 6:130–133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Genetics, University of Wisconsin, 425-G Henry Mall, Madison, Wisconsin, 53706, USA

    Thérèse C. Rytz, Marcus J. Miller & Richard D. Vierstra

  2. Department of Biology, Washington University in St. Louis, Campus Box 1137, One Brookings Drive, St. Louis, Missouri, 63130, USA

    Thérèse C. Rytz & Richard D. Vierstra

Authors
  1. Thérèse C. Rytz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marcus J. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Richard D. Vierstra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard D. Vierstra .

Editor information

Editors and Affiliations

  1. INBIOMED, San Sebastian, Spain

    Manuel S. Rodriguez

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Rytz, T.C., Miller, M.J., Vierstra, R.D. (2016). Purification of SUMO Conjugates from Arabidopsis for Mass Spectrometry Analysis. In: Rodriguez, M. (eds) SUMO. Methods in Molecular Biology, vol 1475. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6358-4_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6358-4_18

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6356-0

  • Online ISBN: 978-1-4939-6358-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation