Saha Kusumika, Yang Jae-Won, Hofmaier Tina, Venkatesan SanthoshKannan, Steinkellner Thomas, Kudlacek Oliver, Sucic Sonja, Freissmuth Michael, Sitte Harald H
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, Paris, France.
Front Physiol. 2021 May 10;12:671034. doi: 10.3389/fphys.2021.671034. eCollection 2021.
The eukaryotic endocytic pathway regulates protein levels available at the plasma membrane by recycling them into specific endosomal compartments. ARFGAP1 is a component of the coat protein I (COPI) complex but it also plays a role in promoting adapter protein-2 (AP-2) mediated endocytosis. The excitatory amino acid transporter-3 (EAAT3) mediates the reuptake of glutamate from the synaptic cleft to achieve rapid termination of synaptic transmission at glutamatergic synapses. In this study, we identified two interacting proteins of EAAT3 by mass spectrometry (MS) ARFGAP1 and ARF6. We explored the role of ARFGAP1 and ARF6 in the endocytosis of EAAT3. Our data revealed that ARFGAP1 plays a role in the recycling of EAAT3, by utilizing its GTPase activating protein (GAP) activity and ARF6 acting as the substrate. ARFGAP1 promotes cargo sorting of EAAT3 a single phenylalanine residue (F508) located at the C-terminus of the transporter. ARFGAP1-promoted AP-2 dependent endocytosis is abolished upon neutralizing F508. We utilized a heterologous expression system to identify an additional motif in the C-terminus of EAAT3 that regulates its endocytosis. Impairment in endocytosis did not affect somatodendritic targeting in cultured hippocampal neurons. Our findings support a model where endocytosis of EAAT3 is a multifactorial event regulated by ARFGAP1, occurring the C-terminus of the transporter, and is the first study to examine the role of ARFGAP1 in the endocytosis of a transport protein.
真核细胞内吞途径通过将蛋白质循环至特定的内体区室来调节质膜上可用的蛋白质水平。ARFGAP1是衣被蛋白I（COPI）复合体的一个组成部分，但它在促进衔接蛋白-2（AP-2）介导的内吞作用中也发挥作用。兴奋性氨基酸转运体-3（EAAT3）介导谷氨酸从突触间隙的重摄取，以实现谷氨酸能突触处突触传递的快速终止。在本研究中，我们通过质谱（MS）鉴定出EAAT3的两种相互作用蛋白——ARFGAP1和ARF6。我们探究了ARFGAP1和ARF6在EAAT3内吞作用中的作用。我们的数据显示，ARFGAP1通过利用其GTP酶激活蛋白（GAP）活性并以ARF6作为底物，在EAAT3的循环利用中发挥作用。ARFGAP1促进EAAT3的货物分选，该转运体C末端有一个单一的苯丙氨酸残基（F508）。中和F508后，ARFGAP1促进的AP-2依赖性内吞作用被消除。我们利用异源表达系统在EAAT3的C末端鉴定出另一个调节其内吞作用的基序。内吞作用受损并不影响培养的海马神经元中树突棘的靶向定位。我们的研究结果支持一种模型，即EAAT3的内吞作用是一个由ARFGAP1调节的多因素事件，发生在转运体的C末端，并且是首个研究ARFGAP1在转运蛋白内吞作用中作用的研究。
