Advertisement

Metabolons on the Plant ER

  • Verena KriechbaumerEmail author
  • Stanley W. Botchway
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1691)

Abstract

Metabolons are protein complexes that contain all the enzymes necessary for a metabolic pathway but also scaffolding proteins. Such a structure allows efficient channeling of intermediate metabolites from one active site to the next and is highly advantageous for labile or toxic intermediates. Here we describe two methods currently used to identify metabolons via protein–protein interaction methodology: immunoprecipitations using GFP-Trap®_A beads to find novel interaction partners and potential metabolon components and FRET-FLIM to test for and quantify protein–protein interactions in planta.

Key words

Endoplasmic reticulum Metabolon Enzymes Protein complex Protein–protein interaction Co-immunoprecipitation GFP-trap FRET-FLIM 

Notes

Acknowledgements

This work was supported by the Science and Technology Facilities Council Program (grant no. 14230008) awarded to Prof. Chris Hawes. 

References

  1. 1.
    Hawes C, Kiviniemi P, Kriechbaumer V (2015) The endoplasmic reticulum: a dynamic and well-connected organelle. J Integr Plant Biol 57:50–62CrossRefPubMedGoogle Scholar
  2. 2.
    Jørgensen K, Rasmussen AV, Morant M, Nielsen AH, Bjarnholt N, Zagrobelny M, Bak S, Møller BL (2005) Metabolon formation and metabolic channelling in the biosynthesis of plant natural products. Curr Opin Plant Biol 8:280–291CrossRefPubMedGoogle Scholar
  3. 3.
    Srere PA (1987) Complexes of sequential metabolic enzymes. Annu Rev Biochem 56:89–124CrossRefPubMedGoogle Scholar
  4. 4.
    Hoppert M, Mayer F (1999) Principles of macromolecular organization and cell function in bacteria and archaea. Cell Biochem Biophys 31:247–284CrossRefPubMedGoogle Scholar
  5. 5.
    Jarvis P, Chen LJ, Li H, Peto CA, Fankhauser C, Chory J (1998) An Arabidopsis mutant defective in the plastid general protein import apparatus. Science 28:100–103CrossRefGoogle Scholar
  6. 6.
    Werhahn W, Niemeyer A, Jansch L, Kruft V, Schmitz UK, Braun H (2001) Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20. Plant Physiol 125:943–954CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Van den Berg B, Clemons WM, Collinson I, Modis Y, Hartmann E, Harrison SC, Rapoport TA (2004) X-ray structure of a protein conducting channel. Nature 427:36–44CrossRefPubMedGoogle Scholar
  8. 8.
    Horie C, Suzuki H, Sakaguchi M, Mihara K (2003) Targeting and assembly of mitochondrial tail-anchored protein Tom5 to the TOM complex depend on a signal distinct from that of tail-anchored proteins dispersed in the membrane. J Biol Chem 278:41462–41471CrossRefPubMedGoogle Scholar
  9. 9.
    Marusic C, Nuttall J, Buriani G, Lico C, Lombardi R, Baschieri S, Benvenuto E, Frigerio L (2007) Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells. BMC Biotechnol 7:12CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Bassard JE, Richert L, Geerinck J, Renault H, Duval F, Ullmann P, Schmitt M, Meyer E, Mutterer J, Boerjan W, De Jaeger G, Mely Y, Goossens A, Werck-Reichhart D (2012b) Protein-protein and protein-membrane associations in the lignin pathway. Plant Cell 24:4465–4482CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Sparkes I, Tolley N, Aller I, Svozil J, Osterrieder A, Botchway S, Mueller C, Frigerio L, Hawes C (2010) Five plant reticulon isoforms share ER localisation, topology, ER membrane shaping properties. Plant Cell 22:1333–1343CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Winkel-Shirley B (2002) Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Plant Physiol 107:142–149CrossRefGoogle Scholar
  13. 13.
    Bassard JE, Mutterer J, Duval F, Werck-Reichhart D (2012) A novel method for monitoring the localization of cytochromes P450 and other endoplasmic reticulum membrane associated proteins: a tool for investigating the formation of metabolons. FEBS J 279:1576–1583CrossRefPubMedGoogle Scholar
  14. 14.
    Hrazdina G, Wagner GJ (1985) Metabolic pathways as enzyme complexes: evidence for the synthesis of phenylpropanoids and flavonoids on membrane associated enzyme complexes. Arch Biochem Biophys 237:88–100CrossRefPubMedGoogle Scholar
  15. 15.
    Lallemand B, Erhardt M, Heitz T, Legrand M (2013) Sporopollenin biosynthetic enzymes interact and constitute a metabolon localized to the endoplasmic reticulum at tapetum cells. Plant Physiol 162:616–625CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nielsen KA, Tattersall DB, Jones PR, Møller BL (2008) Metabolon formation in dhurrin biosynthesis. Phytochemistry 69:88–98CrossRefPubMedGoogle Scholar
  17. 17.
    Dastmalchi M, Bernards MA, Dhaubhadel S (2016) Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H. Plant J 85:689–706CrossRefPubMedGoogle Scholar
  18. 18.
    Kriechbaumer V, Botchway SW, Hawes C (2016) Localization and interactions between Arabidopsis auxin biosynthetic enzymes in the TAA/YUC-dependent pathway. J Exp Bot 67:4195–4207CrossRefPubMedGoogle Scholar
  19. 19.
    Stafford HA (1974) Possible multienzyme complexes regulating the formation of C6-C3 phenolic compounds and lignins in higher plants. Recent Adv Phytochem 8:53–79CrossRefGoogle Scholar
  20. 20.
    Winkel-Shirley B (2001) Flavonoid biosynthesis: a colorful model for genetics, biochemistry, cell biology and biotechnology. Plant Physiol 126:485–493CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ralston L, Yu O (2006) Metabolons involving plant cytochrome P450. Phytochem Rev 5:459–472CrossRefGoogle Scholar
  22. 22.
    Winkel BS (2004) Metabolic channelling in plants. Annu Rev Plant Biol 55:85–107CrossRefPubMedGoogle Scholar
  23. 23.
    Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 437:55–75CrossRefGoogle Scholar
  24. 24.
    Kriechbaumer V, Botchway SW, Slade SE, Knox K, Frigerio L, Oparka K, Hawes C (2015) Reticulomics: protein-protein interaction studies with two plasmodesmata-localized reticulon family proteins identify binding partners enriched at plasmodesmata, endoplasmic reticulum, and the plasma membrane. Plant Physiol 169:1933–1945PubMedPubMedCentralGoogle Scholar
  25. 25.
    Schoberer J, Botchway SW (2014) Investigating protein-protein interactions in the plant endomembrane system using multiphoton-induced FRET-FLIM. Methods Mol Biol 1209:81–95CrossRefPubMedGoogle Scholar
  26. 26.
    Botchway SW, Scherer KM, Hook S, Stubbs CD, Weston E, Bisby RH, Parker AW (2015) A series of flexible design adaptations to the Nikon E-C1 and E-C2 confocal microscope systems for UV, multiphoton and FLIM imaging. J Microsc 258:68–78CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Biological and Medical SciencesOxford Brookes UniversityOxfordUK
  2. 2.Central Laser FacilityScience and Technology Facilities Council (STFC) Rutherford Appleton LaboratoryDidcotUK

Personalised recommendations