Rab GTPases pp 217-231 | Cite as

Analysis of Connecdenn 1–3 (DENN1A-C) GEF Activity for Rab35

  • Patrick D. Allaire
  • Peter S. McPherson
  • Brigitte RitterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)


Rabs (Ras-related proteins in brain) form the largest family of small GTPases and control numerous aspects of membrane trafficking at multiple cellular sites. Rab GTPases toggle between an inactive GDP-bound state and an active GTP-bound state. Activation of Rab GTPases requires guanine nucleotide exchange factors (GEFs) that interact with inactive GDP-bound Rabs and catalyze the removal of GDP, allowing GTP to bind. The largest single family of GEFs for Rabs is comprised of proteins bearing a DENN (differentially expressed in normal and neoplastic cells) domain. In this chapter we describe a biochemical method that directly measures the exchange activity of DENN domains by monitoring loading of GTP onto a Rab GTPase. Rabs are first purified from bacterial or mammalian sources and are then loaded with GDP. Purified DENN domains or DENN domain-bearing proteins are added in the presence of [35S]GTPγS and the transfer of [35S]GTPγS to the Rab is measured by filtering the reaction over nitrocellulose membranes to trap the Rab and thus the associated [35S]GTPγS.

Key words

C9ORF72 Connecdenn DENN domain DENND Endocytosis Endosome GEF GTPase Guanine nucleotide exchange factor Membrane traffic 


  1. 1.
    Richardson PM, Zon LI (1995) Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16. Oncogene 11:1139–1148PubMedGoogle Scholar
  2. 2.
    Fukui K, Sasaki T, Imazumi K et al (1997) Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins. J Biol Chem 272:4655–4658CrossRefPubMedGoogle Scholar
  3. 3.
    Wada M, Nakanishi H, Satoh A et al (1997) Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins. J Biol Chem 272:3875–3878CrossRefPubMedGoogle Scholar
  4. 4.
    Iwasaki K, Staunton J, Saifee O et al (1997) aex-3 encodes a novel regulator of presynaptic activity in C. elegans. Neuron 18:613–622CrossRefPubMedGoogle Scholar
  5. 5.
    Levivier E, Goud B, Souchet M et al (2001) uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways. Biochem Biophys Res Commun 287:688–695CrossRefPubMedGoogle Scholar
  6. 6.
    Marat AL, Dokainish H, McPherson PS (2011) DENN domain proteins: regulators of Rab GTPases. J Biol Chem 286:13791–13800CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Sato M, Sato K, Liou W et al (2008) Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein. EMBO J 27:1183–1196CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Allaire PD, Ritter B, Thomas S et al (2006) Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis. J Neurosci 26:13202–13212CrossRefPubMedGoogle Scholar
  9. 9.
    Allaire PD, Marat AL, Dall’Armi C et al (2010) The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes. Mol Cell 37:370–382CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Yoshimura S, Gerondopoulos A, Linford A et al (2010) Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors. J Cell Biol 191:367–381CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Marat AL, McPherson PS (2010) The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery. J Biol Chem 285:10627–10637CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Wu X, Bradley MJ, Cai Y et al (2011) Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. Proc Natl Acad Sci U S A 108:18672–18677CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Nookala RK, Langemeyer L, Pacitto A et al (2012) Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open Biol 2:120071CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Zhang G, Jung BP, Ho W et al (2007) Isolation and characterization of LCHN: a novel factor induced by transient global ischemia in the adult rat hippocampus. J Neurochem 101:263–273CrossRefPubMedGoogle Scholar
  15. 15.
    Levine TP, Daniels RD, Gatta AT et al (2013) The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics 29:499–503CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Zhang D, Iyer LM, He F et al (2012) Discovery of Novel DENN proteins: implications for the evolution of eukaryotic intracellular membrane structures and human disease. Front Genet 3:283PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Patrick D. Allaire
    • 1
  • Peter S. McPherson
    • 2
  • Brigitte Ritter
    • 3
    Email author
  1. 1.Department of BiologyUniversity of UtahSalt Lake CityUSA
  2. 2.Department of Neurology and Neurosurgery, Montreal Neurological InstituteMcGill UniversityMontrealCanada
  3. 3.Department of BiochemistryBoston University School of MedicineBostonUSA

Personalised recommendations