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Volume 153, Issue 3

Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Free

Abstract

For osmoprotection, can synthesize glycine betaine from externally supplied choline by the Bet system ( products). The major carrier of choline is the high-affinity, proton-driven, secondary transporter BetT, which belongs to the BCCT family of transporters. Fusion proteins consisting of N-terminal fragments of BetT linked to -galactosidase (LacZ) or alkaline phosphatase (PhoA) were constructed. By analysis of 51 fusion proteins with 37 unique fusion-points, the predictions that BetT comprised 12 membrane-spanning regions and that its N- and C-terminal extensions of about 12 and 180 amino acid residues, respectively, were situated in the cytoplasm were confirmed. This is believed to represent the first experimental examination of the membrane topology of a BCCT family protein. Osmotic upshock experiments were performed with spectinomycin-treated cells that had expressed the wild-type or a mutant BetT protein during growth at low osmolality (160 mosmol kg). The choline transport activity of wild-type BetT increased tenfold when the cells were stressed with 0.4 M NaCl (total osmolality 780 mosmol kg). The peak activity was recorded 5 min after the upshock and higher or lower concentrations of NaCl reduced the activity. Deletions of 1–12 C-terminal residues of BetT caused a gradual reduction in the degree of osmotic activation from ten- to twofold. Mutant proteins with deletion of 18–101 residues displayed a background transport activity, but they could not be osmotically activated. The data showed that the cytoplasmic C-terminal domain of BetT plays an important role in the regulation of BetT activity and that C-terminal truncations can cause BetT to be permanently locked in a low-transport-activity mode.

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Keyword(s): ABC, ATP-binding cassette and XP, 5-bromo-4-chloro-3-indolyl phosphate
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2007-03-01
2024-04-20
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Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Escherichia coli
Microbiology 153, 803 (2007); https://doi.org/10.1099/mic.0.2006/003608-0
/content/journal/micro/10.1099/mic.0.2006/003608-0
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