Glycine transporter dimers: evidence for occurrence in the plasma membrane.
Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.
Bartholomäus
Ingo
I
Department of Neurochemistry, Max Planck Institute for Brain Research, Deutschordenstrasse 46, 60529 Frankfurt, Germany.
Milan-Lobo
Laura
L
Nicke
Annette
A
Dutertre
Sébastien
S
Hastrup
Hanne
H
Jha
Alok
A
Gether
Ulrik
U
Sitte
Harald H
HH
Betz
Heinrich
H
Eulenburg
Volker
V
D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D005998:Glycine / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation
D003432:Cross-Linking Reagents; D006026:Glycoside Hydrolases; D005998:Glycine