Constitutive and regulated endocytosis of the glycine transporter GLYT1b is controlled by ubiquitination.
The glycine transporter GLYT1 regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking of GLYT1 to and from the cell surface is critical for its function. Activation of PKC down-regulates the activity of GLYT1 through a mechanism that has so far remained uncharacterized. Here we show that GLYT1b undergoes fast constitutive endocytosis that is accelerated by phorbol esters. Both constitutive and regulated endocytosis occur through a dynamin 2- and clathrin-dependent pathway, accumulating in the transporter in transferrin-containing endosomes. A chimera with the extracellular and transmembrane domains of the nerve growth factor receptor and the COOH-terminal tail of GLYT1 was efficiently internalized through this clathrin pathway, suggesting the presence of molecular determinants for GLYT1b endocytosis in its COOH-terminal tail. Extensive site-directed mutagenesis in this region of the chimera highlighted the involvement of lysine residues in its internalization. In the context of the full-length transporter, lysine 619 played a prominent role in both the constitutive and phorbol 12-myristate 13-acetate-induced endocytosis of GLYT1b, suggesting the involvement of ubiquitin modification of GLYT1b during the internalization process. Indeed, we show that GLYT1b undergoes ubiquitination and that this process is stimulated by phorbol 12-myristate 13-acetate. In addition, this endocytosis is impaired in an ubiquitination-deficient cell line, further evidence that constitutive and regulated endocytosis of GLYT1b is ubiquitin-dependent. It remains to be determined whether GLYT1b recycling might be affected in pathologies involving alterations to the ubiquitin system, thereby interfering with its influence on inhibitory and excitatory neurotransmission.
Fernández-Sánchez
Enrique
E
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Cientificas, Centro de Investigación en Red de Enfermedades Raras, Madrid 28049, Spain.
Martínez-Villarreal
Jaime
J
Giménez
Cecilio
C
Zafra
Francisco
F
D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008985:Monensin / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D054875:Ubiquitination
D002966:Clathrin; D050487:Glycine Plasma Membrane Transport Proteins; D007476:Ionophores; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C493266:Slc6a9 protein, rat; D014168:Transferrin; D008985:Monensin; D034285:Dynamin II; D008239:Lysine; D013755:Tetradecanoylphorbol Acetate