Fig. 4. Bicarbonate inactivates HT1 kinase by stabilizing HT1 interaction with MPK4/12.

(A) Recombinant CBC1, HT1, and MPK4 proteins were incubated with or without 20 mM NaHCO₃ for 30 min, and in vitro phosphorylation assays were performed. (B) CBC1 and HT1 proteins were incubated with or without 20 mM NaHCO₃ or 20 mM NaCl (− controls) in the presence or absence of MPK4 or MPK12 protein. (C) His-HT1 and GST-MPK4 or GST control proteins were used for in vitro pull-down assays with or without 20 mM NaHCO₃. NaHCO₃ or 20 mM NaCl (− controls) were supplemented in all buffers throughout the pull-down assay procedures including the washing step. (D) In vitro pull-down assays were performed using His-HT1 and GST-MPK3, GST-MPK4, GST-MPK12, or GST-MPK11 proteins. (E) In vitro pull-down assays showed reversibility and were performed using the washing buffers supplemented with NaHCO₃ at the indicated concentrations (0, 2, or 20 mM). (F) In vitro pull-down assays were performed using recombinant HT1 (WT, HT1-G89R, and HT1-R173Q) proteins. (G) In vitro pull-down assays were performed using HT1-R102K isoform. (H) In vitro pull-down assays were performed using HT1-A109V isoform. (I) HT1-FLAG and MPK4-GFP or GFP (control) were transiently expressed in Arabidopsis mesophyll cell protoplasts. Coimmunoprecipitation analyses using polyclonal GFP antibodies were performed, and then, precipitated proteins were detected by immunoblot analyses using monoclonal FLAG or GFP antibodies. The immunoblot images showing MPK4-GFP and GFP bands were from the same single membrane, but exposure times are different as indicated next to the images (2 min for MPK4-GFP and 10 s for GFP control) because the MPK4-GFP expression levels were much lower than those of the GFP control. IP, immunoprecipitation.