Constitutive Endocytosis of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter-3 Requires ARFGAP1.

Kusumika Saha,Thomas Steinkellner,Sonja Sucic,Santhoshkannan Venkatesan,Tina Hofmaier,Oliver Kudlacek,Jae-Won Yang,Michael Freissmuth,Harald H Sitte

doi:10.3389/fphys.2021.671034

Abstract

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 via 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 via the C-terminus of the transporter, and is the first study to examine the role of ARFGAP1 in the endocytosis of a transport protein.

Highlights

Glutamate is taken up into cells by excitatory amino acid transporters (EAATs; Danbolt et al, 2016)
Several protein bands were exclusively presented or highly increased in the hEAAT3-engaged rat brain lysates (RBL) sample compared to those in the samples of excitatory amino acid transporter-3 (EAAT3) proteins without RBL incubation and GFP-RBL complexes (Figure 1A). Each lane of these three samples was excised into 10 pieces for Endocytosis of EAAT3 Requires ARFGAP1 B
We found that ARFGAP1 promotes endocytic sorting of EAAT3 via the C-terminus residue F508

Summary

Introduction

Glutamate is taken up into cells by excitatory amino acid transporters (EAATs; Danbolt et al, 2016). Renal wasting of glutamate and aspartate is the most prominent phenotypic consequence resulting from genetic ablation of EAAT3 in mice (Peghini et al, 1997) This dicarboxylic aminoaciduria is recapitulated by loss of function mutations in humans (Bailey et al, 2011). The fact that the absence of EAAT3 does not increase the susceptibility to seizures is in stark contrast to the phenotypic consequences of ablation of EAAT2 (Tanaka et al, 1997) or of EAAT1 (Watanabe et al, 1999) It can, be rationalized by taking into account the finding that EAAT3 only contributes to a very minor fraction of total forebrain glutamate (Holmseth et al, 2012). Internalization of EAAT2 has been reported to rely on the adapter protein arrestin-2/β-arrestin-1 (Ibáñez et al, 2016)

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Conclusion