title: Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification.
abstract: Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.
authors.affiliation: Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstrasse 42, D-80336 München, Germany.
authors.forename: Katrin V
authors.initials: KV
authors.lastname: Koch
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Reinders
authors.forename: Thi-Hieu
authors.initials: TH
authors.lastname: Ho
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.forename: Ralph
authors.initials: R
authors.lastname: Gräf
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004023:Dictyostelium / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D008869:Microtubule-Associated Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins