The glycine transporter GLYT1 interacts with Sec3, a component of the exocyst complex.
Evidence is accumulating that the glycine transporter GLYT1 regulates NMDA receptor function by modulating the glycine concentration in glutamatergic synapses. In this article, we describe a physical and functional interaction between GLYT1 and the exocyst complex. Through a yeast two-hybrid screen to search for proteins capable of interacting with the intracellular C-terminal tail of GLYT1, we identified a protein that is highly homologous to the human and mouse Sec3 protein, a component of the exocyst complex. Pull-down and immunoprecipitation assays confirmed the physical interaction between the C-terminus of GLYT1 and Sec3. Subsequently, immunofluorescence experiments indicated that Sec3-GFP was partially recruited to the plasma membrane upon coexpression with GLYT1. The interaction of GLYT1 with exocyst components was also observed in the native rat brain since complexes immunoprecipitated from brain extracts with anti-GLYT1 antibodies contained both Sec6 and Sec8. Functional assays revealed that Sec3 increased the transporter capacity of GLYT1, suggesting that the exocyst favors insertion of GLYT1 into the plasma membrane.
Cubelos
Beatriz
B
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain.
Giménez
Cecilio
C
Zafra
Francisco
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D005998:Glycine / Q000378:metabolism / Q000493:pharmacokinetics; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins / Q000145:classification / Q000502:physiology*; D016415:Sequence Alignment / Q000379:methods; D014162:Transfection / Q000379:methods; D014169:Transformation, Bacterial; D014316:Tritium / Q000493:pharmacokinetics; D020798:Two-Hybrid System Techniques
D050487:Glycine Plasma Membrane Transport Proteins; D008565:Membrane Proteins; C493268:SLC6A9 protein, human; D029701:Saccharomyces cerevisiae Proteins; D014316:Tritium; D049452:Green Fluorescent Proteins; D005998:Glycine