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Intracellular Lipid Transport pp 35–46Cite as

Following Anterograde Transport of Phosphatidylserine in Yeast in Real Time

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1949))

Abstract

In order to understand how lipids are sorted between cellular compartments, kinetic assays are required to selectively follow the transport of lipid species in cells. We present here a microfluidics-based protocol to follow the transport of phosphatidylserine (PS) in yeast cells from the site of its synthesis, the endoplasmic reticulum (ER), to downstream compartments, primarily the plasma membrane under our conditions. This assay takes advantage of yeast cells lacking Cho1, the enzyme responsible for PS synthesis. Lyso-PS can be added exogenously and is taken up by the cells and converted to PS. Because acylation of lyso-PS to PS appears to occur at the ER, anterograde transport of PS from the ER can then be followed by fluorescent microscopy using the specific PS reporter C2Lact-GFP. We describe the construction of the required cho1Δ yeast strain and the preparation of lyso-PS. We present an example of the use of this assay to follow the activity of the yeast PS transport proteins Osh6 and Osh7.

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References

  1. Wong LH, Copic A, Levine TP (2017) Advances on the transfer of lipids by lipid transfer proteins. Trends Biochem Sci 42:516–530

    Article  CAS  Google Scholar 

  2. Moser von Filseck J, Vanni S, Mesmin B et al (2015) A phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid gradient between membranes. Nat Commun 6:6671

    Article  Google Scholar 

  3. Kannan M, Lahiri S, Liu LK et al (2017) Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER. J Lipid Res 58:553–562

    Article  CAS  Google Scholar 

  4. Georgiev AG, Johansen J, Ramanathan VD et al (2013) Arv1 regulates PM and ER membrane structure and homeostasis but is dispensable for intracellular sterol transport. Traffic 14:912–921

    Article  CAS  Google Scholar 

  5. Yeung T, Gilbert GE, Shi J et al (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319:210–213

    Article  CAS  Google Scholar 

  6. Andersen MH, Graversen H, Fedosov SN et al (2000) Functional analyses of two cellular binding domains of bovine lactadherin. Biochemistry 39:6200–6206

    Article  CAS  Google Scholar 

  7. Del Vecchio K, Stahelin RV (2018) Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments. J Bioenerg Biomembr 50:1–10

    Article  Google Scholar 

  8. Platre MP, Noack LC, Doumane M et al (2018) A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev Cell 45:465–480 e411

    Article  CAS  Google Scholar 

  9. Maeda K, Anand K, Chiapparino A et al (2013) Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins. Nature 501:257–261

    Article  CAS  Google Scholar 

  10. Moser von Filseck J, Copic A, Delfosse V et al (2015) INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate. Science 349:432–436

    Article  CAS  Google Scholar 

  11. Fairn GD, Hermansson M, Somerharju P et al (2011) Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity. Nat Cell Biol 13:1424–1430

    Article  CAS  Google Scholar 

  12. Atkinson K, Fogel S, Henry SA (1980) Yeast mutant defective in phosphatidylserine synthesis. J Biol Chem 255:6653–6661

    CAS  PubMed  Google Scholar 

  13. Yamaoka Y, Yu Y, Mizoi J et al (2011) PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana. Plant J 67:648–661

    Article  CAS  Google Scholar 

  14. Natarajan P, Wang J, Hua Z et al (2004) Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function. Proc Natl Acad Sci U S A 101:10614–10619

    Article  CAS  Google Scholar 

  15. Riekhof WR, Wu J, Gijon MA et al (2007) Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation. J Biol Chem 282:36853–36861

    Article  CAS  Google Scholar 

  16. Longtine MS, McKenzie A 3rd, Demarini DJ et al (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961

    Article  CAS  Google Scholar 

  17. Janke C, Magiera MM, Rathfelder N et al (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21:947–962

    Article  CAS  Google Scholar 

  18. Gietz D, St Jean A, Woods RA et al (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425

    Article  CAS  Google Scholar 

  19. Looke M, Kristjuhan K, Kristjuhan A (2011) Extraction of genomic DNA from yeasts for PCR-based applications. BioTechniques 50:325–328

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Jackson-Verbavatz and Leon labs for support, A-C Gavin for sharing yeast strains, and the ImagoSeine facility at the Institut Jacques Monod, member of the France-Bio-Imaging national research structure (ANR-10-INBS-04). This work is funded by the French National Research Agency grant ExCHANGE (ANR-16-CE13-0006) and by the CNRS.

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Authors and Affiliations

  1. Institut Jacques Monod, CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France

    Juan Martín D’Ambrosio, Véronique Albanèse & Alenka Čopič

Authors
  1. Juan Martín D’Ambrosio
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  2. Véronique Albanèse
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  3. Alenka Čopič
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Corresponding author

Correspondence to Alenka Čopič .

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Editors and Affiliations

  1. Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d’Azur and CNRS, Valbonne, France

    Guillaume Drin

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D’Ambrosio, J.M., Albanèse, V., Čopič, A. (2019). Following Anterograde Transport of Phosphatidylserine in Yeast in Real Time. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_4

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  • DOI: https://doi.org/10.1007/978-1-4939-9136-5_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9135-8

  • Online ISBN: 978-1-4939-9136-5

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