title: Cell surface turnover of the glutamate transporter GLT-1 is mediated by ubiquitination/deubiquitination.
abstract: The main glutamate transporter in the brain, GLT-1, mediates glutamatergic neurotransmission in both physiological and pathological conditions. GLT-1 activity is controlled by both constitutive and regulated trafficking, and although recent evidence indicates that the turnover of this protein in the plasma membrane is accelerated by protein kinase C via an ubiquitin-dependent process, the mechanisms driving the constitutive trafficking of GLT-1 remain unexplored. Here, we used a heterologous system and primary astrocytes to investigate the turnover of GLT-1 and the role of ubiquitin attachment in this process. We show that GLT-1 is endocytosed constitutively in a clathrin-dependent manner, recycling the transporter into endosomes containing EEA1 and Rab4, a marker of rapidly recycling endosomes, and not Rab11 or Rab7, markers of the slow recycling and late endosomal compartments, respectively. We also show that this process is dependent on ubiquitination, because the inhibitor of the ubiquitin-activating enzyme E1, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester, promotes the retention of GLT-1 at the plasma membrane. Moreover, site-directed mutagenesis demonstrated the involvement of lysines 517 and 526 of GLT-1 in the constitutive internalization of the transporter. The translocation of GLT-1 from the recycling endosomes to the plasma membrane was blocked by LDN-57444, a specific inhibitor to the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1, but not by an inhibitor of the related DUB ubiquitin C-terminal hydrolase-L3, supporting the existence of specific ubiquitination/deubiquitination cycles that ensure the correct concentrations of GLT-1 at the cell surface.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: Martínez-Villarreal
authors.forename: Noemí
authors.initials: N
authors.lastname: García Tardón
authors.forename: Ignacio
authors.initials: I
authors.lastname: Ibáñez
authors.forename: Cecilio
authors.initials: C
authors.lastname: Giménez
authors.forename: Francisco
authors.initials: F
authors.lastname: Zafra
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D002966:Clathrin; D027342:Excitatory Amino Acid Transporter 2