Figure 9.

Intracellular Alkalinization Improves the in Vivo Interaction of VSR with Aleurain in nhx5 nhx6.

(A) BiFC in wild-type protoplasts coexpressing SCYCE-VSR2;1 and Aleu-VYNE and equilibrated with acidic buffer, pH 5. A comparison of the resulting signal, expressed as both number of BiFC bodies and BiFC signal relative to the transformation control (VSR1;1-RFPm) signal, as was done in Figure 8, is shown in (E) and (F) below.

(B) BiFC in nhx5 nhx6 protoplasts coexpressing SCYCE-VSR2;1 and Aleu-VYNE and incubated in alkaline equilibration buffer, pH 7. pH was equilibrated in both the wild type and nhx5 nhx6 with 10 µM nigericin, 60 mM KCl, and 10 mM MES/Bis-Tris-propane, pH 5.0 or 7, similar to the in vivo pH calibration described in Methods and Figure 7.

(C) and (D) Expression of inactivated VSR1;1-mRFP control in the wild-type (C) and nhx5 nhx6 (D) protoplasts shown in (A) and (B), respectively. Bar = 5 μm.

(E) and (F) Quantification of the number of BiFC bodies (E) and quantification of the BiFC signal relative to inactivated VSR1;1-mRFP (F), shown in (A) and (B). Bars are means of 65 protoplasts ± sd. Gray bars indicate control values from unequilibrated wild-type and nhx5 nhx6 protoplasts in Figure 8 and are included for comparison with the equilibrated protoplasts presented here. Protoplasts were incubated with pH buffers for 15 min before imaging. Error bars indicate sd. ***P < 0.001, **P < 0.01, by t test.