Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum
Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum
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DOI：
10.1091/mbc.e18-02-0132
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发表时间：
2019-02-15
影响因子：
3.3
通讯作者：
Mueller-Taubenberger, Annette
中科院分区：
文献类型：
--
作者：
Batsios, Petros;Ishikawa-Ankerhold, Hellen C.;Mueller-Taubenberger, Annette
高度保守的精氨酸- trna -蛋白转移酶（Ate1）介导精氨酸化，这是一种在分子水平上尚不完全了解的翻译后修饰。为了研究精氨酸化是否影响一种简单模式生物盘状盘齿龙（Dictyostelium disideum）的肌动蛋白依赖过程，我们敲除了编码Ate1的基因，并表征了Ate1缺失细胞的表型。通过活细胞显微镜观察肌动蛋白细胞骨架动力学显示与野生型细胞相比有显著变化。Ate1-null细胞在底物附着表面几乎完全缺乏局灶性肌动蛋白粘附位点，仅具有弱粘附。在对叶酸或cAMP的二维趋化性实验中，ate1-null细胞的运动性增加。然而，在三维趋化过程中，涉及更有限的条件，ate1-null细胞的运动性显著降低。活细胞成像显示，gfp标记的Ate1快速迁移到新形成的富含肌动蛋白的突起。通过质谱分析，我们在Dictyostelium最丰富的肌动蛋白异构体中鉴定了四个精氨酸化位点，此外还在其他肌动蛋白异构体和几个肌动蛋白结合蛋白中鉴定了精氨酸化位点。用从ate1-null细胞纯化的肌动蛋白进行的体外聚合实验显示，与野生型肌动蛋白相比，其聚合能力有所降低。我们的数据表明，精氨酸化在细胞骨架活动的调节中起着至关重要的作用。
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.