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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)

Abstract

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 

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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

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