Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum

  • The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometricThe highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.show moreshow less

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Author details:Petros BatsiosORCiDGND, Hellen Christina Ishikawa-AnkerholdGND, Heike RothGND, Michael SchleicherGND, Catherine C. L. Wong, Annette Müller-TaubenbergerORCiDGND
DOI:https://doi.org/10.1091/mbc.E18-02-0132
ISSN:1059-1524
ISSN:1939-4586
Pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed?term=30586322
Title of parent work (English):Molecular biology of the cell : the official publication of the American Society for Cell Biology
Publisher:American Society for Cell Biology
Place of publishing:Bethesda
Publication type:Article
Language:English
Year of first publication:2019
Completion year:2019
Release date:2021/04/07
Volume:30
Issue:4
Page number:14
First page:453
Last Page:466
Funding institution:Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [SFB 914/A07, SFB 914/Z01]
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
DDC classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Peer review:Referiert
Publishing method:Open Access / Hybrid Open-Access