Advertisement

Proteomic Characterization of Golgi Membranes Enriched from Arabidopsis Suspension Cell Cultures

  • Sara Fasmer Hansen
  • Berit Ebert
  • Carsten Rautengarten
  • Joshua L. Heazlewood
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1496)

Abstract

The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.

Key words

Golgi apparatus Density gradient centrifugation Subcellular localization Fluorescent protein 

Notes

Acknowledgments

This work was funded by grants from the Australia Research Council (ARC) to the ARC Centre of Excellence in Plant Cell Walls [CE110001007] and the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. J.L.H. is supported by an ARC Future Fellowship [FT130101165]. S.F.H. was supported by a research grant [VKR023371] from VILLUM FONDEN. We also wish to thank the UC Davis Proteomics Core Facility for sample analysis.

References

  1. 1.
    Morré DJ, Mollenhauer HH (2009) The Golgi apparatus: the first 100 years. Springer, New YorkCrossRefGoogle Scholar
  2. 2.
    Scheller HV, Ulvskov P (2010) Hemicelluloses. Annu Rev Plant Biol 61:263–289CrossRefPubMedGoogle Scholar
  3. 3.
    Harholt J, Suttangkakul A, Scheller HV (2010) Biosynthesis of pectin. Plant Physiol 153:384–395CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Song W, Henquet MGL, Mentink RA et al (2011) N-glycoproteomics in plants: perspectives and challenges. J Proteomics 74:1463–1474CrossRefPubMedGoogle Scholar
  5. 5.
    Rennie EA, Ebert B, Miles GP et al (2014) Identification of a sphingolipid alpha-glucuronosyltransferase that is essential for pollen function in Arabidopsis. Plant Cell 26:3314–3325CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wightman R, Turner S (2010) Trafficking of the plant cellulose synthase complex. Plant Physiol 153:427–432CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ordenes VR, Moreno I, Maturana D et al (2012) In vivo analysis of the calcium signature in the plant Golgi apparatus reveals unique dynamics. Cell Calcium 52:397–404CrossRefPubMedGoogle Scholar
  8. 8.
    McFarlane HE, Watanabe Y, Yang WL et al (2014) Golgi- and trans-Golgi network-mediated vesicle trafficking is required for wax secretion from epidermal cells. Plant Physiol 164:1250–1260CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Morré DJ, Mollenhauer HH (1974) The endomembrane concept: a functional integration of endoplasmic reticulum and Golgi apparatus. In: Robards AW (ed) Dynamic aspects of plant infrastructure. McGraw-Hill, New York, pp 84–137Google Scholar
  10. 10.
    Morré DJ, Mollenhauer HH (1964) Isolation of Golgi apparatus from plant cells. J Cell Biol 23:295–305CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Boevink P, Oparka K, Cruz SS et al (1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J 15:441–447CrossRefPubMedGoogle Scholar
  12. 12.
    Akkerman M, Overdijk EJR, Schel JHN et al (2011) Golgi body motility in the plant cell cortex correlates with actin cytoskeleton organization. Plant Cell Physiol 52:1844–1855CrossRefPubMedGoogle Scholar
  13. 13.
    Dunkley TPJ, Watson R, Griffin JL et al (2004) Localization of organelle proteins by isotope tagging (LOPIT). Mol Cell Proteomics 3:1128–1134CrossRefPubMedGoogle Scholar
  14. 14.
    Nikolovski N, Rubtsov D, Segura MP et al (2012) Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics. Plant Physiol 160:1037–1051CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Parsons HT, Weinberg CS, Macdonald LJ et al (2013) Golgi enrichment and proteomic analysis of developing Pinus radiata xylem by free-flow electrophoresis. PLoS One 8:e84669CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Parsons HT, Christiansen K, Knierim B et al (2012) Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel targets involved in plant cell wall biosynthesis. Plant Physiol 159:12–26CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Parsons HT, González Fernández-Niño SM, Heazlewood JL (2014) Separation of the plant Golgi apparatus and endoplasmic reticulum by free-flow electrophoresis. In: Jorrín Novo JV, Komatsu S, Weckwerth W, Weinkoop S (eds) Plant proteomics: methods and protocols, vol 1072, 2nd edn. Humana Press, New York, pp 527–539CrossRefGoogle Scholar
  18. 18.
    Forsmark A, Rossi G, Wadskog I et al (2011) Quantitative proteomics of yeast post-Golgi vesicles reveals a discriminating role for Sro7p in protein secretion. Traffic 12:740–753CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Gilchrist A, Au CE, Hiding J et al (2006) Quantitative proteomics analysis of the secretory pathway. Cell 127:1265–1281CrossRefPubMedGoogle Scholar
  20. 20.
    Mast S, Peng L, Jordan TW et al (2010) Proteomic analysis of membrane preparations from developing Pinus radiata compression wood. Tree Physiol 30:1456–1468CrossRefPubMedGoogle Scholar
  21. 21.
    Zeng W, Jiang N, Nadella R et al (2010) A glucurono(arabino)xylan synthase complex from wheat contains members of the GT43, GT47, and GT75 families and functions cooperatively. Plant Physiol 154:78–97CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Nikolovski N, Shliaha PV, Gatto L et al (2014) Label-free protein quantification for plant Golgi protein localization and abundance. Plant Physiol 166:1033–1043CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. 24.
    Earley KW, Haag JR, Pontes O et al (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45:616–629CrossRefPubMedGoogle Scholar
  25. 25.
    Gene Ontology Consortium (2004) The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res 32:D258–D261CrossRefGoogle Scholar
  26. 26.
    Heazlewood JL, Verboom RE, Tonti-Filippini J et al (2007) SUBA: the Arabidopsis subcellular database. Nucleic Acids Res 35:D213–D218CrossRefPubMedGoogle Scholar
  27. 27.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedGoogle Scholar
  28. 28.
    May MJ, Leaver CJ (1993) Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiol 103:621–627PubMedPubMedCentralGoogle Scholar
  29. 29.
    Tanz SK, Castleden I, Hooper CM et al (2013) SUBA3: a database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis. Nucleic Acids Res 41:D1185–D1191CrossRefPubMedGoogle Scholar
  30. 30.
    Zybailov B, Mosley AL, Sardiu ME et al (2006) Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res 5:2339–2347CrossRefPubMedGoogle Scholar
  31. 31.
    Ebert B, Rautengarten C, Guo X et al (2015) Identification and characterization of a Golgi-localized UDP-xylose transporter family from Arabidopsis. Plant Cell 27:1218–1227CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Sara Fasmer Hansen
    • 1
    • 2
  • Berit Ebert
    • 3
  • Carsten Rautengarten
    • 3
  • Joshua L. Heazlewood
    • 2
    • 3
  1. 1.Department of Plant and Environmental Sciences, Faculty of ScienceUniversity of CopenhagenFrederiksberg CDenmark
  2. 2.Joint BioEnergy Institute and Physical Biosciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  3. 3.ARC Centre of Excellence in Plant Cell Walls, School of BioSciencesThe University of MelbourneParkvilleAustralia

Personalised recommendations