Advertisement

Proteomics of Endosomal Compartments from Plants Case Study: Isolation of Trans-Golgi Network Vesicles

  • Eunsook Park
  • Georgia DrakakakiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)

Abstract

A detailed understanding of endomembrane processes and their biological roles is vital for a complete picture of plant growth and development; however their highly dynamic nature has complicated comprehensive and rigorous studies so far. Recent pioneering efforts have demonstrated that isolation of vesicles in their native state, paired with a quantitative identification of their cargo, offers a viable and practicable approach for the dissection of endomembrane trafficking pathways. The protocol presented in this chapter describes in detail the isolation of the SYP61 trans-Golgi network vesicles from Arabidopsis. With minor alterations, in a few key parameters, it can be adopted to yield a universal procedure for the broad spectrum of plant vesicles.

Key words

Vesicle isolation Immunoisolation Trans-Golgi network (TGN) SYP61 Endomembrane trafficking Proteomics 

Notes

Acknowledgements

This work was supported by the UCD Hellman Fellowship and the NSF-IOS 1258135 to GD.

References

  1. 1.
    Surpin M, Raikhel N (2004) Traffic jams affect plant development and signal transduction. Nat Rev Mol Cell Biol 5:100–109PubMedCrossRefGoogle Scholar
  2. 2.
    Worden N, Park E, Drakakaki G (2012) Trans-Golgi network: an intersection of trafficking cell wall components. J Integr Plant Biol 54: 875–886PubMedGoogle Scholar
  3. 3.
    Hicks GR, Raikhel NV (2010) Advances in dissecting endomembrane trafficking with small molecules. Curr Opin Plant Biol 13:706–713PubMedCrossRefGoogle Scholar
  4. 4.
    Brandizzi F, Fricker M, Hawes C (2002) A greener world: the revolution in plant bioimaging. Nat Rev Mol Cell Biol 3:520–530PubMedCrossRefGoogle Scholar
  5. 5.
    Sparkes I, Brandizzi F (2012) Fluorescent protein-based technologies: shedding new light on the plant endomembrane system. Plant J 70:96–107PubMedCrossRefGoogle Scholar
  6. 6.
    Geldner N, Denervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178PubMedCrossRefGoogle Scholar
  7. 7.
    Drakakaki G, Robert S, Szatmari A-M, Brown MQ, Nagawa S, Van Damme D, Leonard M, Yang Z, Girke T, Schmid SL, Russinova E, Friml J, Raikhel NV, Hicks GR (2011) Clusters of bioactive compounds target dynamic endomembrane networks in vivo. Proc Natl Acad Sci U S A 108:17850–17855PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Kleffmann T, Russenberger D, von Zychlinski A, Christopher W, Sjölander K, Gruissem W, Baginsky S (2004) The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions. Curr Biol 14:354–362PubMedCrossRefGoogle Scholar
  9. 9.
    Mosley AL, Florens L, Wen Z, Washburn MP (2009) A label free quantitative proteomic analysis of the Saccharomyces cerevisiae nucleus. J Proteomics 72:110–120PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Carter C, Pan S, Zouhar J, Avila EL, Girke T, Raikhel NV (2004) The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16:3285–3303PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Eubel H, Heazlewood JL, Millar AH (2006) Isolation and subfractionation of plant mitochondria for proteomic analysis. Methods Mol Biol 355:49–62Google Scholar
  12. 12.
    Eubel H, Meyer EH, Taylor NL, Bussell JD, O’Toole N, Heazlewood JL, Castleden I, Small ID, Smith SM, Millar AH (2008) Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of arabidopsis cell culture peroxisomes. Plant Physiol 148:1809–1829PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Asakura T, Hirose S, Katamine H, Kitajima A, Hori H, Sato MH, Fujiwara M, Shimamoto K, Mitsui T (2006) Isolation and proteomic analysis of rice Golgi membranes: cis-Golgi membranes labeled with GFP-SYP31. Plant Biotechnol 23:475–485CrossRefGoogle Scholar
  14. 14.
    Dunkley TPJ, Hester S, Shadforth IP, Runions J, Weimar T, Hanton SL, Griffin JL, Bessant C, Brandizzi F, Hawes C, Watson RB, Dupree P, Lilley KS (2006) Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci 103:6518–6523PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Nikolovski N, Rubtsov D, Segura MP, Miles GP, Stevens TJ, Dunkley TPJ, Munro S, Lilley KS, Dupree P (2012) Putative glycosyltransferases and other plant golgi apparatus proteins are revealed by LOPIT proteomics. Plant Physiol 160:1037–1051PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Parsons HT, Christiansen K, Knierim B, Carroll A, Ito J, Batth TS, Smith-Moritz AM, Morrison S, McInerney P, Hadi MZ, Auer M, Mukhopadhyay A, Petzold CJ, Scheller HV, Loque D, Heazlewood JL (2012) Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis. Plant Physiol 159:12–26PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Drakakaki G, van de Ven W, Pan S, Miao Y, Wang J, Keinath NF, Weatherly B, Jiang L, Schumacher K, Hicks G, Raikhel N (2012) Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis. Cell Res 22:413–424PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Parsons HT, Drakakaki G, Heazlewood JL (2012) Proteomic dissection of the Arabidopsis Golgi and trans-Golgi network. Front Plant Sci 3:298PubMedCentralPubMedGoogle Scholar
  19. 19.
    Drakakaki G, Zabotina O, Delgado I, Robert S, Keegstra K, Raikhel N (2006) Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development. Plant Physiol 142:1480–1492PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Morris JA, Dorner AJ, Edwards CA, Hendershot LM, Kaufman RJ (1997) Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins. J Biol Chem 272:4327–4334PubMedCrossRefGoogle Scholar
  21. 21.
    da Silva CA, Marty-Mazars D, Bassham DC, Sanderfoot AA, Marty F, Raikhel NV (1997) The syntaxin homolog AtPEP12p resides on a late post-Golgi compartment in plants. Plant Cell 9:571–582CrossRefGoogle Scholar
  22. 22.
    Fonslow BR, Carvalho PC, Academia K, Freeby S, Xu T, Nakorchevsky A, Paulus A, Yates JR (2011) Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT. J Proteome Res 10:3690–3700PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Silva JC, Denny R, Dorschel CA, Gorenstein M, Kass IJ, Li GZ, McKenna T, Nold MJ, Richardson K, Young P, Geromanos S (2005) Quantitative proteomic analysis by accurate mass retention time pairs. Anal Chem 77:2187–2200PubMedCrossRefGoogle Scholar
  24. 24.
    Weatherly DB, Atwood JA 3rd, Minning TA, Cavola C, Tarleton RL, Orlando R (2005) A Heuristic method for assigning a false-discovery rate for protein identifications from Mascot database search results. Mol Cell Proteomics 4:762–772PubMedCrossRefGoogle Scholar
  25. 25.
    Cutillas PR, Vanhaesebroeck B (2007) Quantitative profile of five murine core proteomes using label-free functional proteomics. Mol Cell Proteomics 6:1560–1573PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Plant sciencesUniversity of California DavisDavisUSA

Personalised recommendations