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Trix, a novel Rac guanine-nucleotide exchange factor from Dictyostelium discoideum is an actin-binding protein and accumulates at endosomes
Authors:Strehle Axel  Schleicher Michael  Faix Jan
Institution:1. A. Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, D-80336 München, Germany;2. Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, OE 4350, Carl-Neuberg-Str.1D-30623 Hannover, 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;1. Randall Division of Cell and Molecular Biophysics, King’s College London, SE1 1UL, United Kingdom;2. Institute of Biochemistry, ETH Zurich, Zurich, Switzerland;3. Hannover Medical School, Hannover, Germany;4. Institut für Biochemie und Chemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany;1. School of Chemistry and Environment, College of Food Science and Technology, Shenzhen Institute of Guangdong Ocean University, Guangdong Ocean University, Zhanjiang, 524088, China;2. Southern Marine Science and Engineering Guangdong Laboratory, Zhanjiang, 524025, China;1. Interdisciplinary School of Health Sciences, University of Ottawa, Ottawa, K1N 6N5, Canada;2. Nutrition Research Division, Health Canada, 251 Sir Frederick Banting Driveway, AL2203E, Ottawa, K1A 0K9, Canada;3. School of Nutrition Sciences, University of Ottawa, Ottawa, K1N 6N5, Canada;4. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, K1H 8M5, Canada;5. Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, K1Y 4E9, Canada;1. Laboratory for Single Molecular Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan;2. Laboratory for Cell Signaling Dynamics, Center for Biosystems Dynamics Research, RIKEN, Suita, Osaka, 565-0874, Japan;3. Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan;4. Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., 114 WBSB, Baltimore, MD, 21205, USA;1. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA;2. Howard Hughes Medical Institute, Yale University, New Haven, CT 06520, USA;3. Department of Applied Physics, Yale University, New Haven, CT 06520, USA;4. Department of Physics, Yale University, New Haven, CT 06520, USA;5. Department of Mechanical Engineering and Materials Science, Yale University, New Haven, CT 06520, USA;6. Department of Chemical and Environmental Engineering, Yale University, New Haven, CT 06520, USA;7. Department of Cell Biology, Yale University, New Haven, CT 06520, USA;8. Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06510, USA;1. Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA;2. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY 10032, USA;3. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA;4. Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA;5. Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA;6. Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA;7. Computational Chemistry and Molecular Biophysics Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse – Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA;8. Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA;9. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan;10. Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA;11. Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA;12. Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
Abstract:Small Rho family GTPases are involved in regulation of actin cytoskeleton dynamics. These molecular switches are themselves mainly controlled by specific GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs). We have cloned and initially characterized a novel putative RhoGEF from Dictyostelium discoideum. The predicted 135-kDa protein displays a unique domain organization in its N-terminus by harboring two type3 calponin homology (CH) domains followed by a single type1 CH domain. The C-terminal region encompasses a diffuse B-cell lymphoma homology/pleckstrin homology tandem domain that is typically found in RhoGEFs. We therefore refer to this protein as Trix (triple CH-domain array exchange factor). A recombinant N-terminal region of Trix carrying all three CH domains binds to F-actin and bundles actin filaments. Trix-null mutants are viable and display only subtle defects when compared to wild-type cells with the exception of a substantial decrease in exocytosis of a fluid-phase marker. GFP fusions with the full-length protein or the N-terminal part containing all three CH domains revealed that Trix localizes to the cortical region and strongly accumulates on late endosomes. Our results suggest that Trix is specifically involved in a Rho GTPase-signaling pathway that is required for regulation of the actin cytoskeleton during exocytosis.
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