Transmembrane topology and oligomeric structure of the high-affinity choline transporter

Takashi Okuda, Chieko Osawa, Haruhiko Yamada, Kengo Hayashi, Shizue Nishikawa, Tomoko Ushio, Yuji Kubo, Motoyasu Satou, Haruo Ogawa, Tatsuya Haga

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The high-affinity choline transporter CHT1 mediates choline uptake essential for acetylcholine synthesis in cholinergic nerve terminals. CHT1 belongs to the Na+/glucose cotransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na+/galactose cotransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.

Original languageEnglish
Pages (from-to)42826-42834
Number of pages9
JournalJournal of Biological Chemistry
Volume287
Issue number51
DOIs
Publication statusPublished - 2012 Dec 14

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Topology
Choline
Oligomers
Cysteine
Cells
Galactose
Bacterial Structures
Labeling
Cholinergic Agents
Acetylcholine
Epitopes
HEK293 Cells
Proteins
Monomers
Crystal structure
Molecular weight
Membranes
Scanning
Glucose
Cultured Cells

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Transmembrane topology and oligomeric structure of the high-affinity choline transporter. / Okuda, Takashi; Osawa, Chieko; Yamada, Haruhiko; Hayashi, Kengo; Nishikawa, Shizue; Ushio, Tomoko; Kubo, Yuji; Satou, Motoyasu; Ogawa, Haruo; Haga, Tatsuya.

In: Journal of Biological Chemistry, Vol. 287, No. 51, 14.12.2012, p. 42826-42834.

Research output: Contribution to journalArticle

Okuda, T, Osawa, C, Yamada, H, Hayashi, K, Nishikawa, S, Ushio, T, Kubo, Y, Satou, M, Ogawa, H & Haga, T 2012, 'Transmembrane topology and oligomeric structure of the high-affinity choline transporter', Journal of Biological Chemistry, vol. 287, no. 51, pp. 42826-42834. https://doi.org/10.1074/jbc.M112.405027
Okuda, Takashi ; Osawa, Chieko ; Yamada, Haruhiko ; Hayashi, Kengo ; Nishikawa, Shizue ; Ushio, Tomoko ; Kubo, Yuji ; Satou, Motoyasu ; Ogawa, Haruo ; Haga, Tatsuya. / Transmembrane topology and oligomeric structure of the high-affinity choline transporter. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 51. pp. 42826-42834.
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