The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K+

Abstract

The isolated glycine betaine uptake carrier BetP from Corynebacterium glutamicum was reconstituted in Escherichia coli phospholipid liposomes and its response to osmotic stress studied. The transport activity of BetP, which was previously shown to comprise both osmosensory and osmoregulatory functions, was used to identify the nature of the physicochemical stimulus related to hyperosmotic stress. Putative factors modulating transport activity in response to osmotic stress were dissected. These include type, osmolality and concentration of solutes in the internal and/or external compartment (cationic, anionic, zwitterionic, neutral), as well as membrane strain as a response to increased osmolality. Osmoresponsive activation of BetP was independent of any external factor and of physical alterations of the membrane, but was triggered by a change in the internal K+ concentration. Activation did not depend on the type of anion present and was K+ (or Cs+ and Rb+) specific, as choline and NH(4)+ did not trigger BetP activity. The half-maximal activation of BetP in E.coli phospholipid liposomes was correlated to an internal concentration of 221 +/- 23 mM K+.

Fig. 1. Activity regulation of BetP in dependence of liposomal shrinkage. The uptake of [¹⁴C]glycine betaine in proteoliposomes was measured. (A) In the two experiments, proteoliposomes shrink to an identical extent; however, they differ with respect to an increase in internal K⁺ (circles) or the lack of this increase (squares). The internal buffer contained 76 mM KP_i pH 7.5 (circles) or 76, 38, 25, 19 and 15 mM KP_i pH 7.5, respectively (squares, initial osmolality 200, 100, 67, 50 or 40 mosmol/kg). The lower line connects mean values of two independent experiments using different liposome preparations, whereas the upper curve is combined from five different sets of experiments, performed in duplicate, from 10 different preparations. The external buffer contained 20 mM NaP_i pH 7.5, 25 mM NaCl (100 mosmol/kg) and was adjusted by NaCl to final osmolality values between 100 and 1000 mosmol/kg (circles) or of 200 mosmol/kg (squares). The abscissa indicates the extent of shrinkage (for calculation see Materials and methods). (B) The internal buffer contained 38 mM KP_i pH 7.5 and was adjusted by K⁺-glutamate to an initial osmolality of 100 (circles), 250 (squares, dashed line) or 550 (triangles, dotted line) mosmol/kg. The external buffer contained 20 mM NaP_i pH 7.5, 25 mM NaCl and was adjusted by NaCl to final values between 100 and 1150 mosmol/kg. The values of (identical) external and internal osmolality at the respective starting conditions of the three individual sets of experiments are indicated by vertical lines. Data points represent means of two independent determinations. The calculated final internal K⁺ concentrations, i.e. the concentrations after osmotic up-shift and volume change, reached values between 67.5 and 775 mM (circles), 57 and 655 mM (squares) or 53 and 610 mM (triangles).

Fig. 2. Activity regulation of BetP dependent on internal and external solute composition. The uptake of [¹⁴C]glycine betaine in proteoliposomes was measured. Each line connects mean values of two independent experiments based on two different proteoliposome preparations. Circles: the internal buffer had an initial osmolality of 200 mosmol/kg (76 mM KP_i pH 7.5). The calculated final K⁺ concentration in the different experiments reached values between 67.5 and 675 mM. Squares: the internal buffer had an initial osmolality of 250 mosmol/kg (38 mM KP_i pH 7.5, 150 mM proline); after osmotic up-shift, final K⁺ concentrations between 27 and 270 mM were reached. Triangles: the internal buffer had an initial osmolality of 550 mosmol/kg (38 mM KP_i pH 7.5, 450 mM proline), the final K⁺ concentration reached values between 13.5 and 135 mM. The external buffer contained 20 mM NaPi pH 7.5, 25 mM NaCl. It was adjusted by NaCl (solid symbols) or proline (open circles) to final values between 100 and 1000 mosmol/kg. The experiment indicated by the solid circles is identical to that shown in the upper curve of Figure 1A.

Fig. 3. Activation of BetP is dependent on the internal osmolality. The uptake of [¹⁴C]glycine betaine in proteoliposomes was measured. The values are means of three independent experiments each. (A) The internal buffer contained 38 mM KP_i pH 7.5 (100 mosmol/kg) and was adjusted by KP_i pH 7.5 (solid circles) or proline (open circles) to final values between 100 and 650 or 500 mosmol/kg, respectively. The external buffer contained 20 mM NaP_i pH 7.5, 25 mM NaCl and was adjusted by NaCl to final values between 100 and 650 or 500 mosmol/kg, respectively. (B) The internal buffer (250 mM KP_i pH 7.5) had an initial osmolality of 650 mosmol/kg. The external buffer contained 20 mM NaP_i pH 7.5, 25 mM NaCl and was adjusted by NaCl to final values between 500 and 1000 mosmol/kg. The abscissa indicates the extent of shrinkage (for calculation see Materials and methods).

Fig. 4. Activity regulation of BetP dependent on the type of internal solute. The uptake of [¹⁴C]glycine betaine in proteoliposomes was measured. (A) The internal buffer contained 19 mM KP_i pH 7.5, and 200, 150, 100, 50 or 0 mM proline and was adjusted with KP_i pH 7.5 (circles; 0, 19, 38, 57 or 76 mM), K⁺-glutamate (squares; 0, 25, 50, 75 or 100 mM), choline chloride (triangles; 0, 25, 50, 75 or 100 mM) or glucose (diamonds; 0, 50, 100, 150 or 200 mM) to an initial osmolality of 250 mosmol/kg. The external buffer (20 mM NaP_i pH 7.5, 287.5 mM NaCl) had an osmolality of 625 mosmol/kg. Thus, a constant 2.5-fold shrinkage was obtained in each experiment. The values are means of three independent experiments each. (B) The same data plotted against the calculated final internal cation concentration.

Fig. 5. Activity regulation of BetP dependent on the type of internal cation. The uptake of [¹⁴C]glycine betaine in proteoliposomes was measured. The internal buffer contained 19 mM KP_i pH 7.5, and was adjusted by KP_i pH 7.5 (circles, dashed line; same data as in Figure 1), to an initial internal osmolality of 200 mosmol/kg, or by KCl (squares), RbCl (upward pointing triangles), CsCl (downward pointing triangles), or NH₄Cl (diamonds) to an initial internal osmolality of 250 mosmol/kg. The external buffer contained 20 mM NaP_i pH 7.5, 25 mM NaCl and was adjusted by NaCl (solid symbols) or NH₄Cl (open diamonds) to final values between 200 and 600 mosmol/kg. The external osmolality and the final solute, resp. K⁺ concentration in the liposomal lumen is shown. The external osmolality was adjusted to values between 200 and 500 mosmol/kg. Data points represent means of two independent determinations.

Fig. 6. Determination of the orientation of Strep-tagged BetP in intact cells. (A) Localization of N- and C-terminal domains of Strep-tagged BetP by ELISA. Spheroplasts (open symbols) or inverted membrane vesicles (sonic vesicles, solid symbols) were prepared from isopropyl-β-d-thiogalactopyranoside (IPTG)-induced cells of DH5αmcr pASK-IBA5 betP (circles, N-terminal Strep-tagII), DH5αmcr pASK-IBA3 betP (squares, C-terminal Strep-tagII) and DH5αmcr pASK-IBA5 (triangles, empty vector). The highest absorption of the sonic vesicles derived from each Strep–BetP-expressing strain was set to 100%. Streptavidin–alkaline phosphatase conjugate was used for the immuno detection of Strep–BetP. The amount of spheroplasts and vesicles was standardized as described in the text; the abscissa refers to identical amounts of protein from intact cells. (B) External localization of cysteines at positions 89 and 516 in Cys-less BetP. The proteins were expressed in E.coli and incubated with membrane-impermeable stilbene maleimide (SM). After solubilization and purification, the proteins were incubated with biotin maleimide (BM); the extent of BM reaction with unmodified cysteines was detected by western blotting. The application of equal amounts of BetP protein was controlled by Coomassie Blue staining.