title: Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners.
abstract: Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.
authors.affiliation: From the ‡Proteome Dynamics.
authors.forename: Florian
authors.initials: F
authors.lastname: Paul
authors.affiliation: From the ‡Proteome Dynamics.
authors.forename: Henrik
authors.initials: H
authors.lastname: Zauber
authors.affiliation: §Spatio-Temporal Control of Rho GTPase Signaling.
authors.forename: Laura
authors.initials: L
authors.lastname: von Berg
authors.affiliation: §Spatio-Temporal Control of Rho GTPase Signaling.
authors.forename: Oliver
authors.initials: O
authors.lastname: Rocks
authors.affiliation: ¶Crystallography, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Daumke
authors.affiliation: From the ‡Proteome Dynamics, matthias.selbach@mdc-berlin.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Selbach
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein