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Mol Biol Cell. 2019 Feb 15;30(4):453-466. doi: 10.1091/mbc.E18-02-0132. Epub 2018 Dec 26.

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

Author information

1
Department of Cell Biology (Anatomy III), Ludwig Maximilian University of Munich, 82152 Planegg-Martinsried, Germany.
2
National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

Abstract

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 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.

PMID:
30586322
PMCID:
PMC6594445
DOI:
10.1091/mbc.E18-02-0132
[Indexed for MEDLINE]
Free PMC Article

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