Ubiquitylation-Mediated Control of Polar Auxin Transport: Analysis of Arabidopsis PIN2 Auxin Transport Protein

  • Johannes Leitner
  • Christian LuschnigEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)


Reversible, covalent modification by the small protein ubiquitin acts in a variety of pathways controlling protein fate in virtually all aspects of cellular function. For example, ubiquitylation of plasma membrane proteins modulates their intracellular sorting and turnover, thereby decisively influencing crosstalk between cells and their environment. In recent years, experimental work performed with the model plant Arabidopsis thaliana demonstrated ubiquitylation of a number of plasma membrane proteins, including the auxin efflux carrier protein PIN2. By using solubilized membrane protein immunoprecipitation assays, we established quantitative approaches, suitable for analysis of PIN2 ubiquitylation and variations therein. Applicability of this robust approach is not restricted to PIN auxin carriers, but could be extended to analysis of further plant membrane proteins that are controlled by variations in their ubiquitylation status.

Key words

Membrane protein Ubiquitylation Polar auxin transport Immunoprecipitation PIN2 Arabidopsis 



Establishment of this protocol was supported by a grant from the Austrian Science Funds to C.L. (FWF, P P25931).


  1. 1.
    Göhre V, Spallek T, Haweker H, Mersmann S, Mentzel T, Boller T, de Torres M, Mansfield JW, Robatzek S (2008) Plant pattern-recognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Curr Biol 18(23):1824–1832. doi: 10.1016/j.cub.2008.10.063 PubMedCrossRefGoogle Scholar
  2. 2.
    Abas L, Benjamins R, Malenica N, Paciorek T, Wisniewska J, Moulinier-Anzola JC, Sieberer T, Friml J, Luschnig C (2006) Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat Cell Biol 8(3):249–256. doi: 10.1038/ncb1369 PubMedGoogle Scholar
  3. 3.
    Kasai K, Takano J, Miwa K, Toyoda A, Fujiwara T (2011) High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana. J Biol Chem 286(8):6175–6183. doi:10.1074/jbc.M110.184929PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Roberts D, Pedmale UV, Morrow J, Sachdev S, Lechner E, Tang X, Zheng N, Hannink M, Genschik P, Liscum E (2011) Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3). Plant Cell 23(10):3627–3640. doi: 10.1105/tpc.111.087999 PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Barberon M, Zelazny E, Robert S, Conejero G, Curie C, Friml J, Vert G (2011) Monoubiquitin-dependent endocytosis of the iron-regulated transporter 1 (IRT1) transporter controls iron uptake in plants. Proc Natl Acad Sci U S A 108(32):E450–E458. doi: 10.1073/pnas.1100659108 PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Leitner J, Petrasek J, Tomanov K, Retzer K, Parezova M, Korbei B, Bachmair A, Zazimalova E, Luschnig C (2012) Lysine63-linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth. Proc Natl Acad Sci U S A 109(21):8322–8327. doi: 10.1073/pnas.1200824109 PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Leitner J, Retzer K, Korbei B, Luschnig C (2012) Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots. Plant Signal Behav 7(10):1271–1273. doi: 10.4161/psb.21715 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Reyes FC, Buono R, Otegui MS (2011) Plant endosomal trafficking pathways. Curr Opin Plant Biol 14(6):666–673. doi: 10.1016/j.pbi.2011.07.009 PubMedCrossRefGoogle Scholar
  9. 9.
    Abas L, Luschnig C (2010) Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Anal Biochem 401(2):217–227. doi: 10.1016/j.ab.2010.02.030 PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Kahana A, Gottschling DE (1999) DOT4 links silencing and cell growth in Saccharomyces cerevisiae. Mol Cell Biol 19(10):6608–6620PubMedCentralPubMedGoogle Scholar
  11. 11.
    Wang H, Matsuzawa A, Brown SA, Zhou J, Guy CS, Tseng PH, Forbes K, Nicholson TP, Sheppard PW, Hacker H, Karin M, Vignali DA (2008) Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. Proc Natl Acad Sci U S A 105(51):20197–20202. doi: 10.1073/pnas.0810461105 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Haughn GW, Somerville C (1986) Sulfonylurea-resistant mutants of Arabidopsis thMiana. Mol Gen Genet 204:430–434CrossRefGoogle Scholar
  13. 13.
    Wu M, Stockley PG, Martin WJ (2002) An improved western blotting technique effectively reduces background. Electrophoresis 23(15):2373–2376. doi: 10.1002/1522-2683(200208)23:15<2373::Aid-Elps2373>3.0.Co;2-W

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Applied Genetics and Cell BiologyUniversity of Natural Resources and Life Sciences, BOKU, WienWienAustria

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