A sensor for intracellular ionic strength

Abstract

Cystathionine-beta-synthase (CBS) domains are found in >4,000 proteins in species from all kingdoms of life, yet their functions are largely unknown. Tandem CBS domains are associated with membrane transport proteins, most notably members of the ATP-binding cassette (ABC) superfamily; voltage-gated chloride channels and transporters; cation efflux systems; and various enzymes, transcription factors, and proteins of unknown function. We now show that tandem CBS domains in the osmoregulatory ABC transporter OpuA are sensors for ionic strength that control the transport activity through an electrostatic switching mechanism. The on/off state of the transporter is determined by the surface charge of the membrane and the internal ionic strength that is sensed by the CBS domains. By modifying the CBS domains, we can control the ionic strength dependence of the transporter: deleting a stretch of C-terminal anionic residues shifts the ionic strength dependence to higher values, whereas deleting the CBS domains makes the system largely independent of ionic strength. We present a model for the gating of membrane transport by ionic strength and propose a new role for CBS domains.

Fig. 1.

The effect of osmotic stress, internal ionic strength, on the transport activity of OpuA. (A) Uptake of [¹⁴C]glycine betaine by OpuA in 30 (▵), 55 (■), 85 (□), 110 (●), or 140 (○) mM KPi, pH 7.0, with or without added KCl as indicated on the x axis. The proteoliposomes were composed of 50 mol % DOPE, 12 mol % DOPC, and 38 mol % DOPG. The lumen of the vesicles, isotonic with the external medium, consisted of 9 mM Na₂-ATP, 9 mM MgSO₄ plus 10 (▵), 30 (■), 60 (), 90 (●), or 120 (○) mM KPi, pH 7.0. (B) The same data plotted as a function of internal ionic strength.

Fig. 2.

Schematic of modifications of CBS domains. (A) Modifications made to the CBS moieties (in orange) linked to the transporter (in gray) are shown. OpuA(PstI) contains two amino acid substitutions, but its osmoregulatory properties are identical to wild-type OpuA (not shown). Details of the modifications and C-terminal sequences are specified in the table. Because the genes encoding the OpuA systems are translationally coupled, the sequence coding for the five C-terminal amino acids (EEENK) of OpuAA and containing the ribosome-binding site for opuABC were not modified. (B) Visualization of the expression and purification of OpuA CBS constructs: lane 1, OpuA(PstI); lane 2, OpuAΔ12; lane 3, OpuAΔ61; lane 4, OpuAΔ119; lane 5, OpuA(PstI); lane 6, OpuAΔ12; lane 7, OpuAΔ61. Vesicles (lane 1–4) and Ni-NTA purified proteins (lane 5–7) were analyzed on a Coomassie brilliant blue-stained SDS/PAGE gel (10% polyacrylamide); a total of 6.5 μg of total membrane protein and 2 μg of purified protein were loaded per lane.

Fig. 3.

In vitro activation profiles of OpuA and derivatives. Uptake of [¹⁴C]glycine betaine by OpuA (●), OpuAΔ12 (○), and OpuAΔ61 (▴) was assayed in 90 mM KPi, pH 7.0, with or without added KCl as indicated on the x axis. The proteoliposomes were composed of 50 mol % DOPE, 44 mol % DOPC, 6 mol % DOPG (A); 50 mol % DOPE, 37 mol % DOPC, 13 mol % DOPG (B); 50 mol % DOPE, 32 mol % DOPC, 18 mol % DOPG (C); 50 mol % DOPE, 25 mol % DOPC, 25 mol % DOPG (D); 50 mol % DOPE, 12 mol % DOPC, 38 mol % DOPG (E); or 50 mol % DOPE, 50 mol % DOPG (F). The ATP-regenerating system was enclosed inside the proteoliposomes. The average rates and standard deviations of at least four time points in the linear range (10–45 sec) of the uptake curve are shown.

Fig. 4.

The effect of anionic lipids on the ionic activation of OpuA and derivatives. The maximal (●) and iso-osmotic activity (○) of OpuA (A), OpuAΔ12 (B), and OpuAΔ61 (C) in proteoliposomes as a function of mol % DOPG. The proteoliposomes were composed of DOPE (50 mol %) DOPC (44–0 mol %) and DOPG (6–50 mol %). Maximal activities were obtained from plots such as those presented in Fig. 3 (each repeated two to three times) and correspond to rates of uptake at 200 mM KCl (I_in = ≈0.6); iso-osmotic activities correspond to 0 KCl (I_in = ≈0.2). C Inset shows the data for OpuAΔ119.

Fig. 5.

In vivo activation profiles of OpuA and derivatives. L. lactis Opu401 cells carrying pNZOpuAHis, pNZOpuA(Δ12)His, or pNZOpuA(Δ61)His were grown in M17 supplemented with 0.5% glucose/5 μg/ml chloramphenicol. For the induction of OpuA (●), OpuAΔ12 (○), and OpuAΔ61 (▴), 1.3·10⁻⁴% (vol/vol) culture supernatant of the nisin A producing strain NZ9700 was used. After induction, the cells were washed twice with ice-cold 50 mM Hepes, pH 7.3. Before initiation of transport, cells at 0.4 mg of total protein/ml were preenergized for 5 min with 10 mM glucose (at 30°C). Uptake of [¹⁴C]glycine betaine was assayed in 50 mM Hepes, pH 7.3, supplemented with 50 μg/ml chloramphenicol and 10 mM glucose, with or without added sucrose as indicated on the x axis.

Fig. 6.

Schematic representation of the activities of OpuA, OpuAΔ12, and OpuAΔ61 at low (18 mol %) and high (38 mol %) DOPG. “On” and “off” refer to the active and inactive state of transporter; the “off” state is represented by dotted lines. The ionic strength (salt) dependencies of the on/off switch are indicated. Low I_in refers to 0 KCl (as in Fig. 3) and corresponds to an internal ionic strength of ≈0.2. High I_in refers to 200 mM KCl, yielding an I_in ≈0.6, and intermediate I_in corresponds to a value of ≈0.4. The transporter (ligand-binding receptor, translocator including ABC) is depicted by the gray cylinder, the CBS moiety is in orange-blue (blue depicts the cationic surface possibly interacting with the anionic membrane), and the anionic C terminus is in red; the curled tail in Δ61 depicts the truncated CBS domain; anionic and neutral lipids are represented by red and gray headgroups, respectively. The attraction of the cationic CBS surface and the repulsion of the anionic C terminus by the anionic membrane are highlighted by blue and red arrows, respectively.