Skip to main content
SpringerLink
Log in

Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay

  • Protocol
  • First Online:
Book cover The ESCRT Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1998))

  • 1095 Accesses

Abstract

The endosomal sorting complex required for transport (ESCRT) machinery comprises five complexes that act sequentially to recruit and cluster transmembrane cargo proteins (ESCRT-0), drive membrane curving (ESCRT-I and II), catalyze fission of cargo-containing vesicles (ESCRT-III and VPS/VTA1), and disassemble and recycle the ESCRT-III complex (VPS/VTA1). Since interactions between ESCRT components and cellular or microbial proteins are typically weak, transient, and involve membrane proteins, they are often difficult to study by standard technologies. Here, we describe the utility of high-throughput protein-fragment complementation assays based on the reconstitution of a split luciferase reporter to screen for interactions between any protein and a library of ESCRT proteins in mammalian cells and provide a detailed protocol for these assays.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Henne William M, Buchkovich Nicholas J, Emr Scott D (2011) The ESCRT pathway. Dev Cell 21(1):77–91. https://doi.org/10.1016/j.devcel.2011.05.015

    Article  CAS  PubMed  Google Scholar 

  2. Votteler J, Sundquist Wesley I (2013) Virus budding and the ESCRT pathway. Cell Host Microbe 14(3):232–241. https://doi.org/10.1016/j.chom.2013.08.012

    Article  CAS  PubMed  Google Scholar 

  3. Mehra A, Zahra A, Thompson V, Sirisaengtaksin N, Wells A, Porto M, Köster S, Penberthy K, Kubota Y, Dricot A, Rogan D, Vidal M, Hill DE, Bean AJ, Philips JA (2013) Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog 9(10):e1003734. https://doi.org/10.1371/journal.ppat.1003734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hirano S, Kawasaki M, Ura H, Kato R, Raiborg C, Stenmark H, Wakatsuki S (2006) Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting. Nat Struct Mol Biol 13(3):272–277

    Article  CAS  Google Scholar 

  5. Raiborg C, Bache KG, Gillooly DJ, Madshus IH, Stang E, Stenmark H (2002) Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat Cell Biol 4(5):394–398. http://www.nature.com/ncb/journal/v4/n5/suppinfo/ncb791_S1.html

    Article  CAS  Google Scholar 

  6. Parrish J, Gulyas K, Finley R (2006) Yeast two-hybrid contributions to interactome mapping. Curr Opin Biotechnol 17(4):387–393

    Article  CAS  Google Scholar 

  7. Cusick ME, Klitgord N, Vidal M, Hill DE (2005) Interactome: gateway into systems biology. Hum Mol Genet 14:R171–R181. https://doi.org/10.1093/hmg/ddi335

    Article  CAS  PubMed  Google Scholar 

  8. Garbis S, Lubec G, Fountoulakis M (2005) Limitations of current proteomics technologies. J Chromatogr A 1077(1):1–18

    Article  CAS  Google Scholar 

  9. Remy I, Michnick SW (2004) A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways. Methods 32(4):381–388

    Article  CAS  Google Scholar 

  10. Capdevila-Nortes X, López-Hernández T, Ciruela F, Estévez R (2012) A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells. Anal Biochem 423(1):109–118. https://doi.org/10.1016/j.ab.2012.01.022

    Article  CAS  PubMed  Google Scholar 

  11. Remy I, Michnick SW (1999) Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays. Proc Natl Acad Sci U S A 96(10):5394–5399

    Article  CAS  Google Scholar 

  12. Remy I, Michnick SW (2007) Application of protein-fragment complementation assays in cell biology. BioTechniques 42(2):137–145

    Article  CAS  Google Scholar 

  13. Morell M, Ventura S, Avilés FX (2009) Protein complementation assays: approaches for the in vivo analysis of protein interactions. FEBS Lett 583(11):1684–1691. https://doi.org/10.1016/j.febslet.2009.03.002

    Article  CAS  PubMed  Google Scholar 

  14. Barouch-Bentov R, Neveu G, Xiao F, Beer M, Bekerman E, Schor S, Campbell J, Boonyaratanakornkit J, Lindenbach B, Lu A, Jacob Y, Einav S (2016) Hepatitis C virus proteins interact with the endosomal sorting complex required for transport (ESCRT) machinery via ubiquitination to facilitate viral envelopment. MBio 7(6). https://doi.org/10.1128/mBio.01456-16

  15. Remy I, Michnick S (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3(12):977–979

    Article  CAS  Google Scholar 

  16. Cassonnet P, Rolloy C, Neveu G, Vidalain PO, Chnatier T, Pellet J, Jones L, Muller M, Demeret C, Gaud G, Vuillier F, Lotteau V, Tangy F, Favre M, Jacob Y (2011) Benchmarking a luciferase complementation assay for detecting protein complexes. Nat Methods 8(12):990–992

    Article  CAS  Google Scholar 

  17. Tannous BA, Kim D-E, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11(3):435–443. https://doi.org/10.1016/j.ymthe.2004.10.016

    Article  CAS  PubMed  Google Scholar 

  18. Neveu G, Barouch-Bentov R, Ziv-Av A, Gerber D, Jacob Y, Einav S (2012) Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly. PLoS Pathog 8(8):e1002845

    Article  CAS  Google Scholar 

  19. Rual JF, Hirozane Kishikawa T, Hao T, Bertin N, Li S, Dricot A, Li N, Rosenberg J, Lamesch P, Vidalain PO, Clingingsmith T, Hartley J, Esposito D, Cheo D, Moore T, Simmons B, Sequerra R, Bosak S, Doucette-Stamm L, Le Peuch C, Vandenhaute J, Cusick M, Albala J, Hill D, Vidal M (2004) Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 14(10B):2128–2135

    Article  CAS  Google Scholar 

  20. Neveu G, Cassonnet P, Vidalain P-O, Rolloy C, Mendoza J, Jones L, Tangy F, Muller M, Demeret C, Tafforeau L, Lotteau V, Rabourdin Combe C, Travé G, Al D, Hill D, Vidal M, Favre M, Jacob Y (2012) Comparative analysis of virus-host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase. Methods 58(4):349–359

    Article  CAS  Google Scholar 

  21. Hill SJ, Rolland T, Adelmant G, Xia X, Owen MS, Dricot A, Zack TI, Sahni N, Jacob Y, Hao T, McKinney KM, Clark AP, Reyon D, Tsai SQ, Joung JK, Beroukhim R, Marto JA, Vidal M, Gaudet S, Hill DE, Livingston DM (2014) Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage. Genes Dev 28(17):1957–1975. https://doi.org/10.1101/gad.241620.114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sahni N, Yi S, Taipale M, Fuxman Bass JI, Coulombe-Huntington J, Yang F, Peng J, Weile J, Karras GI, Wang Y, Kovács IA, Kamburov A, Krykbaeva I, Lam MH, Tucker G, Khurana V, Sharma A, Liu Y-Y, Yachie N, Zhong Q, Shen Y, Palagi A, San-Miguel A, Fan C, Balcha D, Dricot A, Jordan DM, Walsh JM, Shah AA, Yang X, Stoyanova A, Leighton A, Calderwood MA, Jacob Y, Cusick ME, Salehi-Ashtiani K, Whitesell LJ, Sunyaev S, Berger B, Barabási A-L, Charloteaux B, Hill DE, Hao T, Roth FP, Xia Y, Walhout AJM, Lindquist S, Vidal M (2015) Widespread macromolecular interaction perturbations in human genetic disorders. Cell 161(3):647–660. https://doi.org/10.1016/j.cell.2015.04.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Rina Barouch-Bentov & Shirit Einav

  2. Département de Virologie, Unité de Génétique Moléculaire des Virus ARN (GMVR), Institut Pasteur, Centre national de la recherche scientifique, Université Paris Diderot, Paris, France

    Yves Jacob

  3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Shirit Einav

Authors
  1. Rina Barouch-Bentov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yves Jacob
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shirit Einav
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shirit Einav .

Editor information

Editors and Affiliations

  1. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

    Emmanuel Culetto

  2. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

    Renaud Legouis

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Barouch-Bentov, R., Jacob, Y., Einav, S. (2019). Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9492-2_21

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9491-5

  • Online ISBN: 978-1-4939-9492-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation