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    • Nystatin Throughout the last few decades evidence has accumu

    2021-11-30

    Throughout the last few decades, evidence has accumulated indicating that NTTs, whose primary location is the cell surface, are subject to a series of regulatory processes of intracellular traffic to and from the membrane (Robinson and Jackson, 2016; Vaughan and Foster, 2013). In addition, lateral mobility and microdomain association within the membrane are critical for the kinetics of neurotransmitter removal, therefore shaping and fine-tuning synaptic transmission (Adkins et al., 2007; Murphy-Royal et al., 2015). All these trafficking events are dynamically controlled by the presence of their respective specific substrates or inhibitors, as well as by diverse posttranslational modifications and interactions with neighboring proteins and Nystatin (Chi and Reith, 2003; Kahlig and Galli, 2003; Murphy-Royal et al., 2015; Saunders et al., 2000; Zahniser and Sorkin, 2009). Despite their differences in mechanism and localization, GLT-1 and DAT share some regulatory features such as sensitivity to protein kinase C signaling (Kahlig et al., 2004; Kalandadze et al., 2002), which promotes ubiquitination-dependent internalization of the transporter mediated by the ubiquitin ligase Nedd4.2 (Garcia-Tardon et al., 2012; Sorkina et al., 2006). Moreover, they share a common localization in lipid rafts on the plasma membrane (Cremona et al., 2011). Diverse pathologies and situations of drug abuse can influence these trafficking events, disturbing the normal function of transporters. Understandably, clarification of all these regulatory mechanisms, including the identification of interacting proteins, has been the objective of many laboratories for the past few years. Regarding characterization of the GLT-1 and DAT interactomes, several specific partners have been identified that regulate diverse aspects of the trafficking process (Robinson and Jackson, 2016; Sager and Torres, 2011). These identifications were mainly based on immunoprecipitation techniques and the yeast two-hybrid system. However, transient or weak protein-protein interactions are frequently refractory to these approaches, thus a number of techniques have been developed that are capable of detecting these types of interactions. One such technique is BioID (proximity-dependent biotin identification), an assay based on the ability of a promiscuous bacterial biotin ligase (BirA R118G, or BirA*) to biotinylate proteins that are permanently or transiently located in the vicinity of the bait protein (Roux et al., 2012, 2013). In a previous report we identified privative members of the GLT-1 interactome by applying the BioID technique to a BirA*-GLT-1 fusion protein, with unfused BirA* as a control to avoid false positives (Piniella et al., 2018). In addition, it was reasoned that a control containing BirA* fused to an unrelated protein transporter, like DAT, might help to discard false positives, although the list had to be carefully scrutinized since it could contain real interactors responsible for the common properties between both transporters. Therefore, the three constructs were used to generate stable transfectants in the neuronal cell line HT22, and proteins labelled in the presence of biotin were isolated with streptavidin-sepharose beads, and characterized by LC-MS/MS. Among others, we found that GLT-1 activity was controlled by regulators of the cytoskeletal dynamics such as the GTPase Rac1 or the effector of CDC42, Borg4, as well as by the βγ subunits of the heterotrimeric G-proteins (Piniella et al., 2018). Interestingly, the latter is also a regulator of DAT, by controlling both the dopamine uptake and reversal of the transporter (Garcia-Olivares et al., 2013, 2017). The transporter-specific interactors of GLT-1 and DAT are analyzed in detail elsewhere (Piniella et al., 2018) Martinez-Blanco et al., unpublished).
    Materials and methods
    Results Previous studies using BioID technique allowed the identification of a number of potential interactors for GLT-1 (73 proteins) and for DAT (23 proteins) (Piniella et al., 2018). Moreover, analysis of the hits yielded 10 proteins shared by both BirA*-GLT-1 and BirA*-DAT (Table 1). However, most of them were discarded from consideration as they were only represented by a single peptide, or were “sticky” nuclear/ribosomal proteins that commonly bind non-specifically to the resin beads used for purification. Thus, we focused on two membrane proteins: Tmem263, a small transmembrane protein with no known function in the nervous system; and Kv7.3, a neuronal protein that forms heterooligomers with the closely related Kv7.2. The voltage-activated potassium channels formed by Kv7.2/7.3 represent the molecular correlate of the M-currents, implicated in controlling the resting membrane potential and spiking activity in neurons.