Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

^*Department of Neurochemistry, Max Planck Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt am Main, Germany; and ^‡Department of Pharmacology, Biocenter of the Johann Wolfgang Goethe University, Marie-Curie Strasse 9, D-60439 Frankfurt am Main, Germany

Edited by H. Ronald Kaback, University of California, Los Angeles, CA, and approved December 11, 2000 (received for review July 14, 2000)

Abstract

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 [³⁵S]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.

Footnotes

↵ † M.H. and A.N. contributed equally to this work.
↵ § To whom reprint requests should be addressed. E-mail: betz{at}mpih-frankfurt.mpg.de.
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.041329498.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.041329498
Abbreviations:

BN,

blue native;

DAT,

dopamine transporter;

GlyT,

glycine transporter;

His,

hexahistidyl;

SERT,

serotonin transporter;

NTA,

nitrilotriacetic acid;

ER,

endoplasmic reticulum;

[125I]sulfo-SHPP,

[125I]sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate;

endo H,

endo-N-acetylglucosaminidase H;

PNGase F,

peptide N-glycosidase F