Analysis of Arf GTP‐Binding Protein Function in Cells

Lee Ann Cohen1, Julie G. Donaldson1

1 Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.12
DOI:  10.1002/0471143030.cb1412s48
Online Posting Date:  September, 2010
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Abstract

This unit describes techniques and approaches that can be used to study the functions of the ADP‐ribosylation factor (Arf) GTP‐binding proteins in cells. There are six mammalian Arfs and many more Arf‐like proteins (Arls), and these proteins are conserved in eukaryotes from yeast to humans. Like all GTPases, Arfs cycle between GDP‐bound, inactive and GTP‐bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs) that catalyze GTP binding and hydrolysis, respectively. This unit describes approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells. A simple protocol for measuring activation (GTP‐binding) of specific Arf proteins in cells using a pull‐down assay is also described. Approaches that can be taken to assess function of GEFs and GAPs in cells is described. Curr. Protoc. Cell Biol. 48:14.12.1‐14.12.17. © 2010 by John Wiley & Sons, Inc.

Keywords: Arf; GTP‐binding proteins; guanine nucleotide exchange factors; GTPase‐activating proteins

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Localization and Function of Specific Arf Proteins in Cells
  • Basic Protocol 2: Assessment of GTP‐Bound Arfs
  • Alternate Protocol 1: Assessment of Arf GTP Levels in Transfected Cells
  • Basic Protocol 3: Assessment of Specificity and Function of Arf GEFs and GAPs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Localization and Function of Specific Arf Proteins in Cells

  Materials
  • 1 M HCl ( appendix 2A)
  • 70% ethanol
  • Culture medium: DMEM containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin solution ( appendix 2A)
  • Cells (e.g., HeLa)
  • Mammalian expression plasmid containing Arf construct of interest with an epitope tag (e.g., Fugene, Roche)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Drugs, growth factors, etc.
  • 2% (w/v) formaldehyde solution in PBS
  • PBS/FBS: 10% (v/v) FBS in PBS and 0.02% (w/v) sodium azide
  • Primary antibody against the epitope tag (e.g., for an HAtag, etc.)
  • 10% (v/v) saponin
  • Secondary antibody: Alexa Fluor 488 or 594 conjugated against the species of the primary antibody
  • Fluoromount G
  • Clear nail polish
  • 12‐mm no. 1 round, glass coverslips
  • 65° to 75°C heating block/plate
  • 100‐mm and 15‐cm tissue culture dishes
  • Watchmaker forceps
  • 6‐ and 12‐well tissue culture plates
  • Parafilm
  • 1.2‐mm thick glass slides
  • Small Kimwipes

Basic Protocol 2: Assessment of GTP‐Bound Arfs

  Materials
  • Frozen bacterial stock for GST‐VHS‐GAT expression (Dell'Angelica et al., )
  • LB/Amp (LB plus 100 µg/ml ampicillin; appendix 2A) plates and medium
  • 1 M isopropyl‐beta‐D‐thiogalactopyranoside (IPTG)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • 2 mM EDTA
  • Lysozyme (lyophilized powder)
  • Protease inhibitors (e.g., 1 mg/ml pepstatin, 1 mM leupeptin, 5 mg/ml aproptinin, and 1 mM PMSF)
  • 10% (v/v) Triton X‐100 stock
  • 10 U/µl DNase I
  • 1 mg/ml RNase stock
  • 1 M dithiothreitol (DTT; appendix 2A)
  • Glutathione Sepharose 4B beads (GE Life Sciences)
  • Lysis buffer (see recipe)
  • 5× SDS‐PAGE sample buffer ( appendix 2A)
  • 2× sample buffer without reducing agent
  • 12% polyacrylamide gel or 4% to 20% gradient gel
  • Specific antibodies for detecting Arf proteins in immunoblots
  • 37°C incubator
  • 14‐ml round‐bottom culture tubes
  • Spectrophotometer
  • Refrigerated centrifuge
  • Tube rotator, 4°C
  • 10‐cm tissue culture dish
  • Cell scraper
  • 1.7‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Microcentrifuge spin columns (e.g., Pierce Spin Cups with cellulose acetate filters)
  • Nitrocellulose paper
  • Additional reagents and equipment for SDS‐PAGE gel (unit 6.1) and nitrocellulose transfer (unit 6.2)

Alternate Protocol 1: Assessment of Arf GTP Levels in Transfected Cells

  • Mammalian expression plasmids containing epitope‐tagged wild type (and mutant) Arf proteins (epitope tag on carboxyl terminus)
  • 6‐well culture plates
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Figures

Videos

Literature Cited

Literature Cited
   Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P.J., and Hsu, V.W. 1997. The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPase‐activating protein for ARF1. EMBO J. 16:7305‐7316.
   Bharti, S., Inoue, H., Bharti, K., Hirsch, D.S., Nie, Z., Yoon, H.Y., Artym, V., Yamada, K.M., Mueller, S.C., Barr, V.A., and Randazzo, P.A. 2007. Src‐dependent phosphorylation of ASAP1 regulates podosomes. Mol. Cell Biol. 27:8271‐8283.
   Boman, A.L., Zhang, C., Zhu, X., and Kahn, R.A. 2000. A family of ADP‐ribosylation factor effectors that can alter membrane transport through the trans‐Golgi. Mol. Biol. Cell 11:1241‐1255.
   Brown, F.D., Rozelle, A.L., Yin, H.L., Balla, T., and Donaldson, J.G. 2001. Phosphatidylinositol 4,5‐bisphosphate and Arf6‐regulated membrane traffic. J. Cell Biol. 154:1007‐1017.
   Casanova, J.E. 2007. Regulation of Arf activation: The Sec7 family of guanine nucleotide exchange factors. Traffic 8:1476‐1485.
   Choi, W., Karim, Z.A., and Whiteheart, S.W. 2006. Arf6 plays an early role in platelet activation by collagen and convulxin. Blood 107:3145‐3152.
   Dell'Angelica, E.C., Puertollano, R., Mullins, C., Aguilar, R.C., Vargas, J.D., Hartnell, L.M., and Bonifacino, J.S. 2000. GGAs: A family of ADP ribosylation factor‐binding proteins related to adaptors and associated with the Golgi complex. J. Cell Biol. 149:81‐94.
   Donaldson, J.G. and Jackson, C.L. 2000. Regulators and effectors of the ARF GTPases. Curr. Opin. Cell Biol. 12:475‐482.
   D'Souza‐Schorey, C. and Chavrier, P. 2006. ARF proteins: Roles in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7:347‐358.
   Eyster, C.A., Higginson, J.D., Huebner, R., Porat‐Shliom, N., Weigert, R., Wu, W.W., Shen, R.F., and Donaldson, J.G. 2009. Discovery of new cargo proteins that enter cells through clathrin‐independent endocytosis. Traffic 10:590‐599.
   Inoue, H. and Randazzo, P.A. 2007. Arf GAPs and their interacting proteins. Traffic 8:1465‐1475.
   Morishige, M., Hashimoto, S., Ogawa, E., Toda, Y., Kotani, H., Hirose, M., Wei, S., Hashimoto, A. Yamada, A., Yano, H., Mazaki, Y., Kodama, H., Nio, Y., Manabe, T., Wada, H., Kobayashi, H., and Sabe, H. 2008. GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion. Nat. Cell Biol. 10:85‐92.
   Santy, L.C. and Casanova, J.E. 2001. Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell Biol. 154:599‐610.
   Skippen, A., Jones, D.H., Morgan, C.P., Li, M., and Cockcroft, S. 2002. Mechanism of ADP ribosylation factor‐stimulated phosphatidylinositol 4,5‐bisphosphate synthesis in HL60 cells. J. Biol. Chem. 277:5823‐5831.
   Volpicelli‐Daley, L.A., Li, Y., Zhang, C.J., and Kahn, R.A. 2005. Isoform‐selective effects of the depletion of ADP‐ribosylation factors 1‐5 on membrane traffic. Mol. Biol. Cell 16:4495‐4508.
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