Skip to main content
SpringerLink
Log in

Subcellular Localization of PI3P in Arabidopsis

  • Protocol
  • First Online:
Plant Endosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2177))

Abstract

Phosphatidylinositol-3-phosphate (PI3P) is a signaling phospholipid enriched in the membranes of late endosomes (LE) and vacuoles. PI3P mediates vacuolar and endosomal trafficking through recruiting PI3P-binding effector proteins to the LE. PI3P is produced from phosphatidylinositol by the PI 3-kinase complex containing VACUOLAR PROTEIN SORTING 34 (VPS34). The role of PI3P has been elucidated by using genetically encoded PI3P biosensors. We previously showed that Arabidopsis VPS38, a component of the VPS34 complex, localized at the LE and that VPS38 is essential for proper PI3P distribution in endosomal and vacuolar trafficking routes. In this chapter, we describe methods for microscopic imaging of PI3P using the PI3P biosensor citrine-2 × FYVE and the PI 3-kinase inhibitors.

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Viotti C, Bubeck J, Stierhof YD, Krebs M, Langhans M, van den Berg W, van Dongen W, Richter S, Geldner N, Takano J, Jurgens G, de Vries SC, Robinson DG, Schumacher K (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22(4):1344–1357. https://doi.org/10.1105/tpc.109.072637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Heucken N, Ivanov R (2018) The retromer, sorting nexins and the plant endomembrane protein trafficking. J Cell Sci 131(2). https://doi.org/10.1242/jcs.203695

  3. Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67:309–335. https://doi.org/10.1146/annurev-arplant-043015-112242

    Article  CAS  PubMed  Google Scholar 

  4. Scheuring D, Viotti C, Kruger F, Kunzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K (2011) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23(9):3463–3481. https://doi.org/10.1105/tpc.111.086918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kim SH, Kwon C, Lee JH, Chung T (2012) Genes for plant autophagy: functions and interactions. Mol Cells 34(5):413–423. https://doi.org/10.1007/s10059-012-0098-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ding X, Zhang X, Otegui MS (2018) Plant autophagy: new flavors on the menu. Curr Opin Plant Biol 46:113–121. https://doi.org/10.1016/j.pbi.2018.09.004

    Article  PubMed  Google Scholar 

  7. Heilmann I (2016) Phosphoinositide signaling in plant development. Development 143(12):2044–2055. https://doi.org/10.1242/dev.136432

    Article  CAS  PubMed  Google Scholar 

  8. Munnik T, Nielsen E (2011) Green light for polyphosphoinositide signals in plants. Curr Opin Plant Biol 14(5):489–497. https://doi.org/10.1016/j.pbi.2011.06.007

    Article  CAS  PubMed  Google Scholar 

  9. Noack LC, Jaillais Y (2017) Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants. Curr Opin Plant Biol 40:22–33. https://doi.org/10.1016/j.pbi.2017.06.017

    Article  CAS  PubMed  Google Scholar 

  10. Chung T (2019) How phosphoinositides shape autophagy in plant cells. Plant Sci 281:146–158. https://doi.org/10.1016/j.plantsci.2019.01.017

    Article  CAS  PubMed  Google Scholar 

  11. Simon ML, Platre MP, Assil S, van Wijk R, Chen WY, Chory J, Dreux M, Munnik T, Jaillais Y (2014) A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis. Plant J 77(2):322–337. https://doi.org/10.1111/tpj.12358

    Article  CAS  PubMed  Google Scholar 

  12. Kihara A, Noda T, Ishihara N, Ohsumi Y (2001) Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J Cell Biol 152(3):519–530

    Article  CAS  Google Scholar 

  13. Itakura E, Kishi C, Inoue K, Mizushima N (2008) Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol Biol Cell 19(12):5360–5372. https://doi.org/10.1091/mbc.E08-01-0080

    Article  PubMed  PubMed Central  Google Scholar 

  14. Vermeer JE, van Leeuwen W, Tobena-Santamaria R, Laxalt AM, Jones DR, Divecha N, Gadella TW Jr, Munnik T (2006) Visualization of PtdIns3P dynamics in living plant cells. Plant J 47(5):687–700. https://doi.org/10.1111/j.1365-313X.2006.02830.x

    Article  CAS  PubMed  Google Scholar 

  15. Matsuoka K, Bassham DC, Raikhel NV, Nakamura K (1995) Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells. J Cell Biol 130(6):1307–1318

    Article  CAS  Google Scholar 

  16. Muller J, Mettbach U, Menzel D, Samaj J (2007) Molecular dissection of endosomal compartments in plants. Plant Physiol 145(2):293–304. https://doi.org/10.1104/pp.107.102863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Haas TJ, Sliwinski MK, Martinez DE, Preuss M, Ebine K, Ueda T, Nielsen E, Odorizzi G, Otegui MS (2007) The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19(4):1295–1312. https://doi.org/10.1105/tpc.106.049346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang J, Cai Y, Miao Y, Lam SK, Jiang L (2009) Wortmannin induces homotypic fusion of plant prevacuolar compartments. J Exp Bot 60(11):3075–3083. https://doi.org/10.1093/jxb/erp136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kleine-Vehn J, Leitner J, Zwiewka M, Sauer M, Abas L, Luschnig C, Friml J (2008) Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting. Proc Natl Acad Sci U S A 105(46):17812–17817. https://doi.org/10.1073/pnas.0808073105

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhuang X, Wang H, Lam SK, Gao C, Wang X, Cai Y, Jiang L (2013) A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell 25(11):4596–4615. https://doi.org/10.1105/tpc.113.118307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ronan B, Flamand O, Vescovi L, Dureuil C, Durand L, Fassy F, Bachelot MF, Lamberton A, Mathieu M, Bertrand T, Marquette JP, El-Ahmad Y, Filoche-Romme B, Schio L, Garcia-Echeverria C, Goulaouic H, Pasquier B (2014) A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy. Nat Chem Biol 10(12):1013–1019. https://doi.org/10.1038/nchembio.1681

    Article  CAS  PubMed  Google Scholar 

  22. Bago R, Malik N, Munson MJ, Prescott AR, Davies P, Sommer E, Shpiro N, Ward R, Cross D, Ganley IG, Alessi DR (2014) Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase. Biochem J 463(3):413–427. https://doi.org/10.1042/BJ20140889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Xu N, Gao XQ, Zhao XY, Zhu DZ, Zhou LZ, Zhang XS (2011) Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation. Plant Mol Biol 77(3):251–260. https://doi.org/10.1007/s11103-011-9806-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lee Y, Kim ES, Choi Y, Hwang I, Staiger CJ, Chung YY, Lee Y (2008) The Arabidopsis phosphatidylinositol 3-kinase is important for pollen development. Plant Physiol 147(4):1886–1897. https://doi.org/10.1104/pp.108.121590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fujiki Y, Yoshimoto K, Ohsumi Y (2007) An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination. Plant Physiol 143(3):1132–1139. https://doi.org/10.1104/pp.106.093864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gao C, Luo M, Zhao Q, Yang R, Cui Y, Zeng Y, Xia J, Jiang L (2014) A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 24(21):2556–2563. https://doi.org/10.1016/j.cub.2014.09.014

    Article  CAS  PubMed  Google Scholar 

  27. Kolb C, Nagel MK, Kalinowska K, Hagmann J, Ichikawa M, Anzenberger F, Alkofer A, Sato MH, Braun P, Isono E (2015) FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 167(4):1361–1373. https://doi.org/10.1104/pp.114.253377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Phan NQ, Kim SJ, Bassham DC (2008) Overexpression of Arabidopsis sorting nexin AtSNX2b inhibits endocytic trafficking to the vacuole. Mol Plant 1(6):961–976. https://doi.org/10.1093/mp/ssn057

    Article  CAS  PubMed  Google Scholar 

  29. Pourcher M, Santambrogio M, Thazar N, Thierry AM, Fobis-Loisy I, Miege C, Jaillais Y, Gaude T (2010) Analyses of sorting nexins reveal distinct retromer-subcomplex functions in development and protein sorting in Arabidopsis thaliana. Plant Cell 22(12):3980–3991. https://doi.org/10.1105/tpc.110.078451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Barberon M, Dubeaux G, Kolb C, Isono E, Zelazny E, Vert G (2014) Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis. Proc Natl Acad Sci U S A 111(22):8293–8298. https://doi.org/10.1073/pnas.1402262111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lee HN, Zarza X, Kim JH, Yoon MJ, Kim SH, Lee JH, Paris N, Munnik T, Otegui MS, Chung T (2018) Vacuolar trafficking protein VPS38 is dispensable for autophagy. Plant Physiol 176(2):1559–1572. https://doi.org/10.1104/pp.17.01297

    Article  CAS  PubMed  Google Scholar 

  32. Liu F, Hu W, Vierstra RD (2018) The vacuolar protein sorting-38 subunit of the Arabidopsis phosphatidylinositol-3-kinase complex plays critical roles in autophagy, endosome sorting, and gravitropism. Front Plant Sci 9:781. https://doi.org/10.3389/fpls.2018.00781

    Article  PubMed  PubMed Central  Google Scholar 

  33. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  34. Takac T, Pechan T, Samajova O, Ovecka M, Richter H, Eck C, Niehaus K, Samaj J (2012) Wortmannin treatment induces changes in Arabidopsis root proteome and post-Golgi compartments. J Proteome Res 11(6):3127–3142. https://doi.org/10.1021/pr201111n

    Article  CAS  PubMed  Google Scholar 

  35. Takac T, Pechan T, Samajova O, Samaj J (2013) Vesicular trafficking and stress response coupled to PI3K inhibition by LY294002 as revealed by proteomic and cell biological analysis. J Proteome Res 12(10):4435–4448. https://doi.org/10.1021/pr400466x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Cell and Molecular Biology and Department of Botany, University of Wisconsin-Madison, Madison, WI, USA

    Han Nim Lee

  2. Department of Biological Sciences, Pusan National University, Busan, Republic of Korea

    Hyera Jung & Taijoon Chung

Authors
  1. Han Nim Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hyera Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Taijoon Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Taijoon Chung .

Editor information

Editors and Affiliations

  1. Department of Botany and Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

    Marisa S. Otegui

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lee, H.N., Jung, H., Chung, T. (2020). Subcellular Localization of PI3P in Arabidopsis. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0767-1_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0766-4

  • Online ISBN: 978-1-0716-0767-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation