Proximity Labeling-Based Mapping of the EAAT3 Interactome and Identification of C-terminal Interacting Partners

Abstract ID 129625 Poster Board 139 Plasma membrane neurotransmitter transporters clear neurotransmitters from the extracellular space and play a key role in regulating neuronal signaling. The neuronal excitatory amino acid transporter 3, EAAT3, is essential for glutamate clearance and modulation of glutamatergic tone. EAAT3 also transports cysteine into the cell where it serves as a rate-limiting substrate for the synthesis of glutathione, a key neuronal antioxidant. EAAT3 mutations are associated with several neuropsychiatric disorders such as schizophrenia and obsessive-compulsive disorders that may be due to its role in glutamate or cysteine transport. The activity of plasma membrane transporters can be altered by changes in transport kinetics, expression levels and trafficking to and from the plasma membrane. The C-terminus of EAAT3, specifically the ⁵⁰⁴VNGGF⁵⁰⁸ motif is important for trafficking and the somatodendritic targeting of EAAT3 in hippocampal neurons. We had previously identified proteins interacting with purified GFP-EAAT3 wild-type (WT) and GFP-EAAT3 (504-509 AAAAAA) from rat brain lysates using a mass spectrometry (MS) based proteomics approach. We have now used the proximity labeling technique, TurboID to complement the results of classic affinity purification/MS-based interactome mapping and to capture transient interactions with endogenous proteins. The studies were conducted in the Neuro2A cell line and in primary cortical neuronal cultures. The TurboID tag was fused to EAAT3 WT or C- terminal mutants of EAAT3 and a cytosolic TurboID construct served as a spatial control. The TurboID tag covalently labels EAAT3 adjacent proteins with biotin in a proximity-dependent manner. The biotinylated proteins were enriched using streptavidin beads and identified by label-free mass spectrometry quantification. Pathway analysis of the proximity labeled proteins, identified pathways involved in regulation and signaling by Rho GTPase and membrane trafficking. Our comprehensive analysis of the EAAT3 interactome network will help us gain insights into the regulation and localization of the glutamate transporter in neurons.