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Rab GTPases pp 73-83 | Cite as

Analysis of the Interactions Between Rab GTPases and Class V Myosins

  • Andrew J. Lindsay
  • Stéphanie Miserey-Lenkei
  • Bruno GoudEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)

Abstract

Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli’s Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid “living chip” assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation.

Key words

Myosin V Rab GTPases Protein–protein interactions Yeast two-hybrid Coimmunoprecipitation 

Notes

Acknowledgements

The work of AJL for this publication was supported by a joint Health Research Board/Marie Curie Mobility Fellowship (MCPD/2009/6) and a Science Foundation Ireland Programme Grant (09/IN1/B2629) to Mary W. McCaffrey, UCC. BG was supported by the Institut Curie, the Centre National de la Recherche Scientifique, and the Agence Nationale pour la Recherche (ANR Grant 2010 BLAN 122902).

References

  1. 1.
    Sellers JR (2000) Myosins: a diverse superfamily. Biochim Biophys Acta 1496(1):3–22CrossRefPubMedGoogle Scholar
  2. 2.
    Rudolf R, Bittins CM, Gerdes HH (2011) The role of myosin V in exocytosis and synaptic plasticity. J Neurochem 116(2):177–191. doi: 10.1111/j.1471-4159.2010.07110.x CrossRefPubMedGoogle Scholar
  3. 3.
    Muller T, Hess MW, Schiefermeier N, Pfaller K, Ebner HL, Heinz-Erian P, Ponstingl H, Partsch J, Rollinghoff B, Kohler H, Berger T, Lenhartz H, Schlenck B, Houwen RJ, Taylor CJ, Zoller H, Lechner S, Goulet O, Utermann G, Ruemmele FM, Huber LA, Janecke AR (2008) MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity. Nat Genet 40(10):1163–1165CrossRefPubMedGoogle Scholar
  4. 4.
    McCaffrey MW, Lindsay AJ (2012) Roles for myosin Va in RNA transport and turnover. Biochem Soc Trans 40(6):1416–1420CrossRefPubMedGoogle Scholar
  5. 5.
    Hammer JA 3rd, Sellers JR (2011) Walking to work: roles for class V myosins as cargo transporters. Nat Rev Mol Cell Biol 13(1):13–26CrossRefPubMedGoogle Scholar
  6. 6.
    Kelly EE, Horgan CP, Goud B, McCaffrey MW (2012) The Rab family of proteins: 25 years on. Biochem Soc Trans 40(6):1337–1347CrossRefPubMedGoogle Scholar
  7. 7.
    Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10(8):513–525CrossRefPubMedGoogle Scholar
  8. 8.
    Trybus KM (2008) Myosin V from head to tail. Cell Mol Life Sci 65(9):1378–1389CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Fukuda M, Kuroda TS, Mikoshiba K (2002) Slac2-a/Melanophilin, the Missing Link between Rab27 and Myosin Va: implications of a tripartite protein complex for melanosome transport. J Biol Chem 277(14):12432–12436CrossRefPubMedGoogle Scholar
  10. 10.
    Wu X, Wang F, Rao K, Sellers JR, Hammer JA 3rd (2002) Rab27a is an essential component of melanosome receptor for myosin Va. Mol Biol Cell 13(5):1735–1749CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Wu XS, Rao K, Zhang H, Wang F, Sellers JR, Matesic LE, Copeland NG, Jenkins NA, Hammer JA 3rd (2002) Identification of an organelle receptor for myosin-Va. Nat Cell Biol 4(4):271–278CrossRefPubMedGoogle Scholar
  12. 12.
    Hume AN, Collinson LM, Hopkins CR, Strom M, Barral DC, Bossi G, Griffiths GM, Seabra MC (2002) The leaden gene product is required with Rab27a to recruit myosin Va to melanosomes in melanocytes. Traffic 3(3):193–202CrossRefPubMedGoogle Scholar
  13. 13.
    Wollert T, Patel A, Lee YL, Provance DW Jr, Vought VE, Cosgrove MS, Mercer JA, Langford GM (2011) Myosin5a tail associates directly with Rab3A-containing compartments in neurons. J Biol Chem 286(16):14352–14361CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Roland JT, Kenworthy AK, Peranen J, Caplan S, Goldenring JR (2007) Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3. Mol Biol Cell 18(8):2828–2837CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Roland JT, Lapierre LA, Goldenring JR (2009) Alternative splicing in class v myosins determines association with rab10. J Biol Chem 284(2):1213–1223CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Lapierre LA, Kumar R, Hales CM, Navarre J, Bhartur SG, Burnette JO, Provance DW Jr, Mercer JA, Bahler M, Goldenring JR (2001) Myosin vb is associated with plasma membrane recycling systems. Mol Biol Cell 12(6):1843–1857CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B (2013) Identification and characterization of multiple novel Rab-myosin Va interactions. Mol Biol Cell 24(21):3420–3434. doi: 10.1091/mbc.E13-05-0236 CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Janoueix-Lerosey I, Jollivet F, Camonis J, Marche PN, Goud B (1995) Two-hybrid system screen with the small GTP-binding protein Rab6. Identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6. J Biol Chem 270(24):14801–14808CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Andrew J. Lindsay
    • 1
  • Stéphanie Miserey-Lenkei
    • 2
  • Bruno Goud
    • 2
    Email author
  1. 1.Molecular Cell Biology Laboratory, School of Biochemistry and Cell Biology, Biosciences InstituteUniversity College CorkCorkIreland
  2. 2.Molecular Mechanisms of Intracellular Transport, Unité Mixte de Recherche 144, Centre National de la Recherche ScientifiqueInstitut CurieParisFrance

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