Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes.
Na(+)/Cl(-)-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [(35)S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na(+)/Cl(-)-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.
Horiuchi
M
M
Department of Neurochemistry, Max Planck Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt am Main, Germany.
Nicke
A
A
Gomeza
J
J
Aschrafi
A
A
Schmalzing
G
G
Betz
H
H
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D005998:Glycine