title: The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus.
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.
authors.affiliation: Institut für Biochemie der Universität zu Köln, Zülpicher Strasse 47, 50674 Köln, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schiller
authors.forename: René
authors.initials: R
authors.lastname: Rübenhagen
authors.forename: Reinhard
authors.initials: R
authors.lastname: Krämer
authors.forename: Susanne
authors.initials: S
authors.lastname: Morbach
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000907:Antibodies, Bacterial / Q000737:chemistry; D000918:Antibody Specificity / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001622:Betaine / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002414:Cations, Monovalent / Q000737:chemistry; D003352:Corynebacterium / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005998:Glycine / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D011188:Potassium / Q000737:chemistry*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics; D027981:Symporters; D014882:Water-Electrolyte Balance / Q000235:genetics
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D002352:Carrier Proteins; D002414:Cations, Monovalent; D008081:Liposomes; D010446:Peptide Fragments; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; D001622:Betaine; D011188:Potassium; D005998:Glycine