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PMID: 8991097 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Membrane topology of the high-affinity L-glutamate transporter (GLAST-1) of the central nervous system.

The Journal of cell biology ·Vol. 135 ·No. 6 Pt 2 ·1996-12-00 ·Pages 1867-77

Wahle S, Stoffel W

Abstract

The membrane topology of the high affinity, Na(+)-coupled L-glutamate/L-aspartate transporter (GLAST-1) of the central nervous system has been determined. Truncated GLAST-1 cDNA constructs encoding protein fragments with an increasing number of hydrophobic regions were fused to a cDNA encoding a reporter peptide with two N-glycosylation sites. The respective cRNA chimeras were translated in vitro and in vivo in Xenopus oocytes. Posttranslational N-glycosylation of the two reporter consensus sites monitors the number, size, and orientation of membrane-spanning domains. The results of our experiments suggest a novel 10-transmembrane domain topology of GLAST-1, a representative of the L-glutamate neurotransmitter transporter family, with its NH2 and COOH termini on the cytoplasmic side, six NH2-terminal hydrophobic transmembrane alpha-helices, and four COOH-terminal short hydrophobic domains spanning the bilayer predicted as beta-sheets.

MeSH Terms
Amino Acid Sequence Amino Acid Transport System X-AG Animals Biological Transport/physiology Carrier Proteins/analysis,chemistry,metabolism Cell Membrane/chemistry Central Nervous System/chemistry,metabolism Conserved Sequence Cytoplasm/chemistry Female Glycoproteins/analysis,chemistry,metabolism Glycosylation Molecular Sequence Data Oocytes/chemistry Protein Structure, Secondary Protein Structure, Tertiary Recombinant Fusion Proteins/chemistry,metabolism Xenopus
Chemicals
Amino Acid Transport System X-AG Carrier Proteins Glycoproteins Recombinant Fusion Proteins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Wahle S
Institute of Biochemistry I, Medical Faculty, University of Cologne, Germany.
Stoffel W
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.135.6.1867
Published
1996-12-00
Pages
1867-77
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2133971
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.135.6.1867 PMC Full Text View journal details
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