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

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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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    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, 287(51), 42826-42834. https://doi.org/10.1074/jbc.M112.405027