Osmosensor and Osmoregulator Properties of the Betaine Carrier BetP from Corynebacterium glutamicum in Proteoliposomes

Abstract

The secondary glycine betaine uptake system BetP of Corynebacterium glutamicum was purified from Escherichia coli membranes in strep-tagged form after heterologous expression of the betP gene and was reconstituted in E. coli lipids. BetP retained its kinetic properties (V max and K m for betaine and Na ؉ ) as compared with intact cells. The influence of driving forces (Na ؉ gradient and/or electrical potential) on betaine uptake was quantified in proteoliposomes. BetP was effectively regulated by the external osmolality and was stimulated by the local anesthetic tetracaine. A shift of the optimum of osmotic stimulation to higher osmolalities was linearly correlated with an increasing share of phosphatidyl glycerol, the major lipid of the C. glutamicum plasma membrane in the E. coli lipid proteoliposomes. This finding correlates with results demonstrating an identical shift when betP was expressed in E. coli instead of C. glutamicum. These data indicate that (i) BetP comprises all elements of osmosensing and osmoregulatory mechanisms of betaine uptake, (ii) osmoregulation of BetP is directly related to protein/membrane interactions, (iii) the turgor pressure presumably plays no major role in osmoregulation of BetP, and (iv) the regulatory properties of BetP may be related to the physical state of the surrounding membrane.

CaiT of Escherichia coli, a New Transporter Catalyzing l-Carnitine/γ-Butyrobetaine Exchange

“…In this way, CaiT clearly differs from other betaine 3 Available at tcdb.ucsd.edu/tcdb/tcfamilybrowse.php?tcnameϭ2.A.2. transporters (ProP of E. coli and BetP of C. glutamicum), whose activities strongly depend on medium osmolality (23,44). BetP (like CaiT, a member of the BCCT family) possesses N-and C-terminal extensions that are required for osmosensing by the transporter (22).…”

“…Some of these transporters have been shown to utilize electrochemical ion gradients (H ϩ , Na ϩ ) for substrate accumulation. In addition, some members of the family are osmotically regulated and share hydrophilic extensions at the N and/or C termini (19,20,22,23).…”

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…Functional reconstitution of the proteins was performed as described (61). Briefly, liposomes (20 mg phospholipid/mL) from E. coli polar lipid extract phospholipids (Avanti Polar Lipids) were prepared by extrusion through polycarbonate filters (400-nm pore size) and diluted 1:4 in buffer (250 mM KPi, pH 7.5).…”

“…Cell-culture and protein-preparation methods were described previously (61). Uptake of [ 14 C]-betaine was measured in E. coli MKH13 cells (62).…”

Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP

“…Protein Reconstitution into Liposomes. Functional reconstitution of BetP and its mutants were performed as described (50). Briefly, liposomes (20 mg phospholipid/mL) from E. coli polar lipids (Avanti polar lipids) were prepared by extrusion through polycarbonate filters (100 nm pore-size) and diluted 1∶4 in buffer (250 mM KPi or 250 mM Tris-HCl, pH 7.5).…”