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Chemical Genomics Screening for Biomodulators of Endomembrane System Trafficking

  • Carlos Rubilar-Hernández
  • Glenn R. Hicks
  • Lorena NorambuenaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)

Abstract

Cell proteins traffic through complex and tightly regulated pathways. Although the endomembrane system is essential, its different pathways are still not well understood. In order to dissect protein trafficking pathways, chemical genomic screenings have been performed. This strategy has been utilized to successfully discover bioactive chemicals with a specific cellular action and in most cases, tunable and reversible effects. Once the bioactive chemical is identified, further strategies can be used to find the target proteins that are important for functionality of trafficking pathways. This approach can be combined with the powerful genetic tools available for model organisms. Drug-hypersensitive and drug-resistant mutant isolation can lead to the identification of cellular pathways affected by a bioactive chemical and reveal its protein target(s). Here, we describe an approach to look for hypersensitive and resistant mutants to a specific bioactive chemical that affects protein trafficking in yeast. This approach can be followed and adapted to any other pathway or cellular process that can be screened phenotypically, serving as a guide for novel screens in yeast. More importantly, information provided by this approach can potentially be extrapolated to other organisms like plants. Thus, the method described can be of broad utility to plant biologists.

Key words

Bioactive compound Carboxypeptidase Y Chemical biology Endomembrane Endocytosis Primary and secondary screening Secretory route 

Notes

Acknowledgements

This work is supported by grant FONDECYT1120289 (LN, CRH) and National Science Foundation Grant MCB-0515963 (GRH).

References

  1. 1.
    Mishev K, Dejonghe W, Russinova E (2013) Small molecules for dissecting endomembrane trafficking: a cross-systems view. Chem Biol 20: 475–486PubMedCrossRefGoogle Scholar
  2. 2.
    Zouhar J, Rojo E (2009) Plant vacuoles: where did they come from and where are they heading? Curr Opin Plant Biol 12:677–684PubMedCrossRefGoogle Scholar
  3. 3.
    Xiang L, Etxeberria E, Van den Ende W (2013) Vacuolar protein sorting mechanisms in plants. FEBS J 280:979–993PubMedCrossRefGoogle Scholar
  4. 4.
    Bassham D, Raikhel N (2000) Plant are not just green yeast. Plant Physiol 122:999–1001PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Sanderfoot A, Assaad F, Raikhel N (2000) The Arabidopsis genome. an abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors. Plant Physiol 124:1558–1569PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Rojo E, Zouhar J, Kovaleva V, Hong S, Raikhel NV (2003) The AtC–VPS protein complex is localized to the tonoplast and the prevacuolar compartment in Arabidopsis. Mol Biol Cell 14:361–369PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Zouhar J, Hicks G, Raikhel N (2004) Sorting inhibitors (sortins): chemical compounds to study vacuolar sorting in Arabidopsis. Proc Natl Acad Sci U S A 101:9497–9501PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Norambuena L, Zouhar J, Hicks G, Raikhel N (2008) Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae. BMC Chem Biol 8:1PubMedCentralPubMedGoogle Scholar
  9. 9.
    Lokey RS (2003) Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia. Curr Opin Chem Biol 7:91–96PubMedCrossRefGoogle Scholar
  10. 10.
    Schreiber S (2005) Small molecules: the missing link in the central dogma. Nat Chem Biol 1:64–66PubMedGoogle Scholar
  11. 11.
    Hicks G, Raikhel N (2012) Small molecules present large opportunities in plant biology. Annu Rev Plant Biol 63:261–282PubMedCrossRefGoogle Scholar
  12. 12.
    Eggert U (2013) The why and how of phenotypic small-molecule screens. Nat Chem Biol 9:206–209PubMedGoogle Scholar
  13. 13.
    Rojas-Pierce M, Titapiwatanakun B, Sohn E, Fang F, Larive C, Blakeslee J, Cheng Y, Cuttler S, Peer W, Murphy A, Raikhel N (2007) Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin. Chem Biol 14:1366–1376PubMedCrossRefGoogle Scholar
  14. 14.
    Robert S, Chary S, Drakakaki G, Li S, Yang Z, Raikhel N, Hicks G (2008) Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1. Proc Natl Acad Sci U S A 105:8464–8469PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Drakakaki G, Robert S, Szatmari A, Brown M, Nagawa S, van Damme D, Leonard M, Yang Z, Schmid S, Russinova E, Friml J, Raikhel N, Hicks G (2011) Clusters of bioactive compounds target dynamic endomembrane networks in vivo. Proc Natl Acad Sci U S A 108:17850–17855PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Rosado A, Hicks G, Norambuena L, Rogachev I, Meir S, Pourcel L, Zouhar J, Brown M, Boirsdore M, Puckrin R, Cutler S, Rojo E, Aharoni A, Raikhel N (2011) Sortin1-hypersensitive mutants link vacuolar-trafficking defects and flavonoid metabolism in Arabidopsis vegetative tissues. Chem Biol 18:187–197PubMedCrossRefGoogle Scholar
  17. 17.
    Park S, Fung P, Nishimura N, Jensen D, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow T, Alfred S, Bonetta D, Finkelstein R, Provart N, Desveaux D, Rodriguez P, McCourt P, Zhu J, Schroeder J, Volkman B, Cutler S (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324: 1068–1071PubMedCentralPubMedGoogle Scholar
  18. 18.
    De Rybel B, Audenaert D, Vert G, Rozhon W, Mayerhofer J, Peelman F, Coutuer S, Denayer T, Jansen L, Nguyen L, Vanhoutte I, Beemster G, Vleminckx K, Jonak C, Chory J, Inzé D, Russinova E, Beeckman T (2009) Chemical inhibition of a subset of arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling. Chem Biol 16:594–604PubMedCrossRefGoogle Scholar
  19. 19.
    Stearns T, Hoyt M, Botstein D (1990) Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Gen 124:251–262Google Scholar
  20. 20.
    Chan T, Carvalho J, Riles L, Zheng X (2000) A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR). Proc Natl Acad Sci U S A 97:13227–13232PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Butcher R, Schreiber S (2004) Identification of Ald6p as the target of a class of small-molecule suppressors of FK506 and their use in network dissection. Proc Natl Acad Sci U S A 101: 7868–7873PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Giaever G, Flaherty P, Kumm J, Proctor M, Nislow C, Jaramillo D, Chu A, Jordan M, Arkin A, Davis R (2004) Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc Natl Acad Sci U S A 101:793–798PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Hoon S, Onge R, Giaever G, Nislow C (2008) Yeast chemical genomics and drug discovery: an update. Trends Pharmacol Sci 29:499–504PubMedCrossRefGoogle Scholar
  24. 24.
    Kemmer D, McHardy L, Hoon S, Delphine Rebérioux D, Giaever G, Nislow C, Roskelley C, Roberge M (2009) Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis. BMC Microbiol 9:9PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792PubMedCrossRefGoogle Scholar
  26. 26.
    Ruepp A, Zollner A, Maier D, Albermann K, Hani J, Mokrejs M, Tetko I, Güldener U, Mannhaupt G, Münsterkötter M, Mewes HW (2004) The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. Nucleic Acids Res 32:5539–5545PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Baudin A, Ozier-Kalogeropoulos O, Denouel A, Lacroute F, Cullin C (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Wach A, Brachat A, Pöhlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808PubMedCrossRefGoogle Scholar
  29. 29.
    Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936–4948PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Carlos Rubilar-Hernández
    • 1
  • Glenn R. Hicks
    • 2
  • Lorena Norambuena
    • 1
    Email author
  1. 1.Plant Molecular Biology Laboratory, Department of Biology, Faculty of SciencesUniversity of ChileSantiagoChile
  2. 2.Department of Botany and Plant Sciences, Center for Plant Cell BiologyUniversity of CaliforniaRiversideUSA

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