Carboxyl terminus of the glycine transporter GLYT1 is necessary for correct processing of the protein.
The high affinity glycine transporter in neurons and glial cells is the primary means of inactivating synaptic glycine. To understand the structure-function relationships, especially the role of the intracellular carboxyl- and amino-terminal domains, we have modified the glycine transporter GLYT1 by using a polymerase chain reaction-based mutagenesis approach. Deletion of the first 30 amino acids of the amino terminus does not alter transport of glycine. Truncation of the last 34 amino acids of the carboxyl terminus did not impair glycine transport, but progressively more extensive deletions produced a progressive decrease in transport activity. All the fully active or partially active forms of the transporter retain the characteristic sodium and chloride dependence of the wild type. When the nonfunctional mutants of the carboxyl terminus were examined by an immunofluorescence technique, the carrier was no longer found in the membrane. This suggests that the carboxyl terminus of GLYT1 may be involved in the membrane insertion process. Moreover, the transporter that is not fully processed is not functional, because transport activity cannot be rescued in a solubilization-reconstitution experiment.
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Spain.
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L
Olivares
C
C
Aragón
C
C
Giménez
F
F
Zafra
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000096:biosynthesis; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins; D015151:Immunoblotting; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D017931:DNA Primers; D050487:Glycine Plasma Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005998:Glycine