Skip to main content
SpringerLink
Log in

Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates

  • Protocol
  • First Online:
Golgi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2557))

Abstract

Rab GTPases are key regulators of membrane trafficking. When GTP-bound, or “active,” Rabs are anchored to membranes and recruit effector proteins that mediate vesicle formation, transport, and fusion. Rabs are inactivated by GTPase-activating proteins (Rab-GAPs), which catalyze GTP hydrolysis, rendering Rabs cytosolic. In vivo, C-terminal prenylation modifications link activated Rabs to organelle and vesicle membranes, yet historically, in vitro Rab-GAP activity assays have been performed in the absence of membranes. We have developed a method for assaying Rab-GAP activity in a physiological context, with dissociation of the Rab from the membrane serving as a readout for Rab-GAP activity. Given that membrane-binding status is a key consequence of Rab activation state, this assay will be useful for the study of a wide range of Rab/Rab-GAP pairs.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 249.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
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

Similar content being viewed by others

Change history

  • 11 February 2023

    The original version of the book was inadvertently published without incorporating the author’s proof corrections mentioned below. The chapters have now been corrected and approved by the author.

References

  1. Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119–149. https://doi.org/10.1152/physrev.00059.2009

    Article  CAS  Google Scholar 

  2. Pfeffer SR (2017) Rab GTPases: master regulators that establish the secretory and endocytic pathways. Mol Biol Cell 28:712–715

    Article  CAS  Google Scholar 

  3. Müller MP, Goody RS (2017) Molecular control of Rab activity by GEFs, GAPs and GDI. Small GTPases 9:5–21. https://doi.org/10.1080/21541248.2016.1276999

    Article  CAS  Google Scholar 

  4. Lamber EP, Siedenburg A-C, Barr FA (2019) Rab regulation by GEFs and GAPs during membrane traffic. Curr Opin Cell Biol 59:34–39. https://doi.org/10.1016/j.ceb.2019.03.004

    Article  CAS  Google Scholar 

  5. Eberth A, Ahmadian MR (2009) In vitro GEF and GAP assays. Curr Protoc Cell Biol 43:14.9.1–14.9.25. https://doi.org/10.1002/0471143030.cb1409s43

    Article  Google Scholar 

  6. Gomes AQ, Ali BR, Ramalho JS et al (2003) Membrane targeting of Rab GTPases is influenced by the prenylation motif. Mol Biol Cell 14:1882–1899. https://doi.org/10.1091/mbc.e02-10-0639

    Article  CAS  Google Scholar 

  7. Wu Y-W, Tan K-T, Waldmann H et al (2007) Interaction analysis of prenylated Rab GTPase with Rab escort protein and GDP dissociation inhibitor explains the need for both regulators. Proc Natl Acad Sci 104:12294–12299. https://doi.org/10.1073/pnas.0701817104

    Article  CAS  Google Scholar 

  8. Thomas LL, Fromme JC (2016) GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis. J Cell Biol 215:499–513. https://doi.org/10.1083/jcb.201608123

    Article  CAS  Google Scholar 

  9. Du L-L, Novick P (2001) Yeast Rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p. Mol Biol Cell 12:1215–1226. https://doi.org/10.1091/mbc.12.5.1215

    Article  CAS  Google Scholar 

  10. Pan X, Eathiraj S, Munson M, Lambright DG (2006) TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442:303–306. https://doi.org/10.1038/nature04847

    Article  CAS  Google Scholar 

  11. Thomas LL, Highland CM, Fromme JC (2021) Arf1 orchestrates Rab GTPase conversion at the trans-Golgi network. Mol Biol Cell 32:1104–1120. https://doi.org/10.1091/mbc.E20-10-0664

    Article  CAS  Google Scholar 

  12. Olson F, Hunt CA, Szoka FC et al (1979) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta 557:9–23. https://doi.org/10.1016/0005-2736(79)90085-3

    Article  CAS  Google Scholar 

  13. Mui B, Chow L, Hope MJ (2003) Extrusion technique to generate liposomes of defined size. In: Methods in enzymology. Academic Press, pp 3–14

    Google Scholar 

  14. Klemm RW, Ejsing CS, Surma MA et al (2009) Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network. J Cell Biol 185:601–612. https://doi.org/10.1083/jcb.200901145

    Article  CAS  Google Scholar 

  15. Lachmann J, Barr FA, Ungermann C (2012) The Msb3/Gyp3 GAP controls the activity of the Rab GTPases Vps21 and Ypt7 at endosomes and vacuoles. Mol Biol Cell 23:2516–2526. https://doi.org/10.1091/mbc.E11-12-1030

    Article  CAS  Google Scholar 

  16. John Peter AT, Lachmann J, Rana M et al (2013) The BLOC-1 complex promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3. J Cell Biol 201:97–111. https://doi.org/10.1083/jcb.201210038

    Article  CAS  Google Scholar 

  17. Bigay J, Gounon P, Robineau S, Antonny B (2003) Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature 426:563–566. https://doi.org/10.1038/nature02108

    Article  CAS  Google Scholar 

  18. Bigay J, Casella J-F, Drin G et al (2005) ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J 24:2244–2253. https://doi.org/10.1038/sj.emboj.7600714

    Article  CAS  Google Scholar 

  19. Ha VL, Thomas GMH, Stauffer S, Randazzo PA (2005) Preparation of myristoylated Arf1 and Arf6. In: GTPases regulating membrane dynamics. Academic Press, pp 164–174

    Google Scholar 

  20. Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature 443:651–657. https://doi.org/10.1038/nature05185

    Article  CAS  Google Scholar 

  21. van Meer G, de Kroon AIPM (2011) Lipid map of the mammalian cell. J Cell Sci 124:5–8. https://doi.org/10.1242/jcs.071233

    Article  CAS  Google Scholar 

  22. Bigay J, Antonny B (2012) Curvature, lipid packing, and electrostatics of membrane organelles: defining cellular territories in determining specificity. Dev Cell 23:886–895. https://doi.org/10.1016/j.devcel.2012.10.009

    Article  CAS  Google Scholar 

  23. Goody RS, Hofmann-Goody W (2002) Exchange factors, effectors, GAPs and motor proteins: common thermodynamic and kinetic principles for different functions. Eur Biophys J 31:268. https://doi.org/10.1007/s00249-002-0225-3

    Article  CAS  Google Scholar 

Download references

Author Contributions

C.M.H.: Writing (original draft; review and editing), Methodology (optimization), Investigation, Funding acquisition. L.L.T.: Writing (review and editing), Methodology (conceptualization, optimization), Investigation. J.C.F. Writing (review and editing), Methodology (conceptualization), Funding acquisition, Supervision.

Author information

Authors and Affiliations

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

    Carolyn M. Highland, Laura L. Thomas & J. Christopher Fromme

  2. HHMI and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Laura L. Thomas

Authors
  1. Carolyn M. Highland
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura L. Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Christopher Fromme
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Christopher Fromme .

Editor information

Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Yanzhuang Wang

  2. Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Vladimir V Lupashin

  3. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Todd R. Graham

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Highland, C.M., Thomas, L.L., Fromme, J.C. (2023). Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates. In: Wang, Y., Lupashin, V.V., Graham, T.R. (eds) Golgi. Methods in Molecular Biology, vol 2557. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2639-9_29

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2639-9_29

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2638-2

  • Online ISBN: 978-1-0716-2639-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation