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Analysis of Arf1 GTPase-Dependent Membrane Binding and Remodeling Using the Exomer Secretory Vesicle Cargo Adaptor

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The Golgi Complex

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1496))

Abstract

Protein–protein and protein–membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest.

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References

  1. Gillingham AK, Munro S (2007) The small G proteins of the Arf family and their regulators. Annu Rev Cell Dev Biol 23:579–611

    Article  CAS  PubMed  Google Scholar 

  2. Donaldson JG, Jackson CL (2011) ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol 12:362–375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cherfils J (2014) Arf GTPases and their effectors: assembling multivalent membrane-binding platforms. Curr Opin Struct Biol 29C:67–76

    Article  Google Scholar 

  4. Wang CW, Hamamoto S, Orci L, Schekman R (2006) Exomer: a coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast. J Cell Biol 174:973–983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Heldwein EE, Macia E, Wang J, Yin HL, Kirchhausen T, Harrison SC (2004) Crystal structure of the clathrin adaptor protein 1 core. Proc Natl Acad Sci U S A 101:14108–14113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Paczkowski JE, Richardson BC, Strassner AM, Fromme JC (2012) The exomer cargo adaptor structure reveals a novel GTPase-binding domain. EMBO J 31:4191–4203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sanchatjate S, Schekman R (2006) Chs5/6 complex: a multiprotein complex that interacts with and conveys chitin synthase III from the trans-Golgi network to the cell surface. Mol Biol Cell 17:4157–4166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Trautwein M, Schindler C, Gauss R, Dengjel J, Hartmann E, Spang A (2006) Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi. EMBO J 25:943–954

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ziman M, Chuang JS, Tsung M, Hamamoto S, Schekman R (1998) Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae. Mol Biol Cell 9:1565–1576

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Barfield RM, Fromme JC, Schekman R (2009) The exomer coat complex transports Fus1p to the plasma membrane via a novel plasma membrane sorting signal in yeast. Mol Biol Cell 20:4985–4996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Aridor M, Fish KN, Bannykh S, Weissman J, Roberts TH, Lippincott-Schwartz J, Balch WE (2001) The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly. J Cell Biol 152:213–229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lee MC, Orci L, Hamamoto S, Futai E, Ravazzola M, Schekman R (2005) Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell 122:605–617

    Article  CAS  PubMed  Google Scholar 

  13. Beck R, Sun Z, Adolf F, Rutz C, Bassler J, Wild K, Sinning I, Hurt E, Brugger B, Bethune J, Wieland F (2008) Membrane curvature induced by Arf1-GTP is essential for vesicle formation. Proc Natl Acad Sci U S A 105:11731–11736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Krauss M, Jia JY, Roux A, Beck R, Wieland FT, De Camilli P, Haucke V (2008) Arf1-GTP-induced tubule formation suggests a function of Arf family proteins in curvature acquisition at sites of vesicle budding. J Biol Chem 283:27717–27723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Boucrot E, Pick A, Camdere G, Liska N, Evergren E, McMahon HT, Kozlov MM (2012) Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149:124–136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lundmark R, Doherty GJ, Vallis Y, Peter BJ, McMahon HT (2008) Arf family GTP loading is activated by, and generates, positive membrane curvature. Biochem J 414:189–194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kahn RA, Randazzo P, Serafini T, Weiss O, Rulka C, Clark J, Amherdt M, Roller P, Orci L, Rothman JE (1992) The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport. J Biol Chem 267:13039–13046

    CAS  PubMed  Google Scholar 

  18. Goldberg J (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95:237–248

    Article  CAS  PubMed  Google Scholar 

  19. Randazzo PA, Terui T, Sturch S, Fales HM, Ferrige AG, Kahn RA (1995) The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch. J Biol Chem 270:14809–14815

    Article  CAS  PubMed  Google Scholar 

  20. McMahon HT, Gallop JL (2005) Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438:590–596

    Article  CAS  PubMed  Google Scholar 

  21. Stachowiak JC, Brodsky FM, Miller EA (2013) A cost-benefit analysis of the physical mechanisms of membrane curvature. Nat Cell Biol 15:1019–1027

    Article  CAS  PubMed  Google Scholar 

  22. Rockenbauch U, Ritz AM, Sacristan C, Roncero C, Spang A (2012) The complex interactions of Chs5p, the ChAPs, and the cargo Chs3p. Mol Biol Cell 23:4402–4415

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Richardson BC, Fromme JC (2015) Biochemical methods for studying kinetic regulation of Arf1 activation by Sec7. Methods Cell Biol 130:101–126

    Article  PubMed  PubMed Central  Google Scholar 

  24. Paczkowski JE, Fromme JC (2014) Structural basis for membrane binding and remodeling by the exomer secretory vesicle cargo adaptor. Dev Cell 30:610–624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Klemm RW, Ejsing CS, Surma MA, Kaiser HJ, Gerl MJ, Sampaio JL, de Robillard Q, Ferguson C, Proszynski TJ, Shevchenko A, Simons K (2009) Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network. J Cell Biol 185:601–612

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the Fromme lab for helpful discussions. This work was supported by NIH/NIGMS grant R01GM098621.

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, 457 Weill Hall, Ithaca, NY, 14853, USA

    Jon E. Paczkowski & J. Christopher Fromme

  2. Department of Molecular Biology and Howard Hughes Medical Institute, Princeton University, Princeton, NJ, 08544, USA

    Jon E. Paczkowski

Authors
  1. Jon E. Paczkowski
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  2. J. Christopher Fromme
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Corresponding author

Correspondence to J. Christopher Fromme .

Editor information

Editors and Affiliations

  1. Dept of Molecular Biology & Genetics, Cornell University, Ithaca, New York, USA

    William J. Brown

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Paczkowski, J.E., Fromme, J.C. (2016). Analysis of Arf1 GTPase-Dependent Membrane Binding and Remodeling Using the Exomer Secretory Vesicle Cargo Adaptor. In: Brown, W. (eds) The Golgi Complex. Methods in Molecular Biology, vol 1496. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6463-5_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6463-5_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6461-1

  • Online ISBN: 978-1-4939-6463-5

  • eBook Packages: Springer Protocols

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