title: A pull-down procedure for the identification of unknown GEFs for small GTPases.
abstract: Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Koch
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Amrita
authors.initials: A
authors.lastname: Rai
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Imtiaz
authors.initials: I
authors.lastname: Ali
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bleimling
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Timon
authors.initials: T
authors.lastname: Friese
authors.affiliation: b Department of Chemical Biology , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Brockmeyer
authors.affiliation: b Department of Chemical Biology , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Janning
authors.affiliation: c Institut Curie, PSL Research University, CNRS UMR 144 , Paris , France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Goud
authors.affiliation: d Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München , Garching , Germany.
authors.forename: Aymelt
authors.initials: A
authors.lastname: Itzen
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Müller
authors.affiliation: a Department of Structural Biochemistry , Max Planck Institute of Molecular Physiology , Dortmund , Germany.
authors.forename: Roger S
authors.initials: RS
authors.lastname: Goody
mesh_terms: D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C064456:SEC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: GEF; enrichment; in vitro transcription/translation; nucleotide exchange factor; pull-down; small GTPase; yeast two-hybrid