title: Calnexin-assisted biogenesis of the neuronal glycine transporter 2 (GlyT2).
abstract: The neuronal transporter GlyT2 is a polytopic, 12-transmembrane domain, plasma membrane glycoprotein involved in the removal and recycling of synaptic glycine from inhibitory synapses. Mutations in the human GlyT2 gene (SLC6A5) that cause deficient glycine transport or defective GlyT2 trafficking are the second most common cause of hyperekplexia or startle disease. In this study we examined several aspects of GlyT2 biogenesis that involve the endoplasmic reticulum chaperone calnexin (CNX). CNX binds transiently to an intermediate under-glycosylated transporter precursor and facilitates GlyT2 processing. In cells expressing GlyT2, transporter accumulation and transport activity were attenuated by siRNA-mediated CNX knockdown and enhanced by CNX overexpression. GlyT2 binding to CNX was mediated by glycan and polypeptide-based interactions as revealed by pharmacological approaches and the behavior of GlyT2 N-glycan-deficient mutants. Moreover, transporter folding appeared to be stabilized by N-glycans. Co-expression of CNX and a fully non-glycosylated mutant rescues glycine transport but not mutant surface expression. Hence, CNX discriminates between different conformational states of GlyT2 displaying a lectin-independent chaperone activity. GlyT2 wild-type and mutant transporters were finally degraded in the lysosome. Our findings provide further insight into GlyT2 biogenesis, and a useful framework for the study of newly synthesized GlyT2 transporters bearing hyperekplexia mutations.
authors: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
authors: Esther
authors: E
authors: Arribas-González
authors: Pablo
authors: P
authors: Alonso-Torres
authors: Carmen
authors: C
authors: Aragón
authors: Beatriz
authors: B
authors: López-Corcuera
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D037281:Calnexin / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D006031:Glycosylation; D007700:Kinetics; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D059748:Proteolysis; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D056811:Unfolded Protein Response
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; C493269:Slc6a5 protein, rat; D014415:Tunicamycin; D037281:Calnexin; D019284:Thapsigargin; D005959:Glucosidases; D008361:Mannosidases
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