Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification |
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| Authors: | Koch Katrin V Reinders Yvonne Ho Thi-Hieu Sickmann Albert Gräf Ralph |
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| Institution: | 1. Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstrasse 42, D-80336 München, Germany;2. Protein Mass Spectrometry and Functional Proteomics Group Rudolf-Virchow-Center for Experimental Biomedicine Versbacher Str. 9, D-97078 Würzburg, Germany;1. Ludwig-Maximilians-Universitaet Muenchen, Institut fuer Zellbiologie (ABI), Schillerstr. 42, D-80336 Muenchen, Germany;2. University of Turin, Department of Clinical and Biological Sciences, I-10043 Orbassano, Italy;3. From the Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and;4. the Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904;1. Department of Health and Education, Universidade Estadual do Norte do Paraná, 86360-000 Paraná, Brazil;2. Universidade Federal de Mato Grosso do Sul, 79400-000 Coxim, Mato Grosso do Sul, Brazil;3. Department of Morphological Sciences, Universidade Estadual de Maringá, 87020-900 Maringá, Paraná, Brazil;1. Department of Cell Biology, FB2, University of Bremen, P.O. Box 33 04 40, 28334 Bremen, Germany;2. Cellular and Molecular Pharmacology, Louvain Drug Research Institute, Université catholique de Louvain, avenue E. Mounier 73 bte B1.73.05, B-1200 Brussels, Belgium;3. Research School of Biology, ANU College of Medicine, Biology and Environment, Canberra, Australia;1. Department of Biology & BK21 – Plus Research Team for Bioactive Control Technology, College of Natural Sciences, Chosun University, Gwangju 501-759, Republic of Korea |
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| 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. |
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