The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus.
Biochemistry
; 43(19): 5583-91, 2004 May 18.
Article
em En
| MEDLINE
| ID: mdl-15134432
ABSTRACT
The glycine betaine carrier BetP of Corynebacterium glutamicum was recently shown to function both as an osmosensor and as an osmoregulator in proteoliposomes by sensing changes in the internal K(+) concentration as a measure of hyperosmotic stress. In vivo analysis of mutants carrying deletions at the C-terminal extension of BetP indicated that this domain participates in osmostress-dependent activity regulation. To address the question, whether a putative K(+) sensor is located within the C-terminal domain, several mutants with truncations in this domain were purified and reconstituted in proteoliposomes of Escherichia coli phospholipids, since this in vitro system allowed variation of the K(+) concentration at the lumenal side. Truncation of 12 amino acids led to a partly deregulated BetP in terms of osmoregulation; however, K(+) sensitivity was not impaired in this mutant. The deletion of 25 amino acid residues at the C-terminal end of BetP led to both deregulation of the carrier activity, i.e., high activity independent of external osmolality, and loss of K(+)-dependent transport stimulation, indicating that this region of the C-terminal domain is necessary for K(+) sensing and/or K(+)-dependent carrier activation. Immunological and proteolysis analyses showed that BetP and its recombinant forms were reconstituted in a right-side-out orientation, i.e., the C-terminal domain faces the lumen of the proteoliposomes and is thus able to detect the K(+) signal at the inside. This is the first experimental demonstration of a direct connection between an osmotic stimulus, i.e., the change in internal K(+), and a putative sensor domain.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Peptídeos
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Potássio
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Proteínas de Bactérias
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Equilíbrio Hidroeletrolítico
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Betaína
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Proteínas de Transporte
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Corynebacterium
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Glicina
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2004
Tipo de documento:
Article
País de afiliação:
Alemanha