Consistent with previous observations, PM H ATPase activity was much higher in vesicles isolated from pks5 1 than in vesicles isolated from Col 0 . Similar results were observed with vesicles isolated from the pks5 6 mutant. PM H ATPase activity in vesicles isolated from the pks5 3 and pks5 4 mutants was much lower than that of the wild type . These results provide further support for a negative correlation between PM H ATPase activity and PKS5 kinase activity. To provide additional evidence that changes inPM H ATPase activity in the pks5 mutants are due to changes in PKS5 kinase activity, we added recombinant PKS5 proteins to transport assays with plasma membrane vesicles isolated from the pks5 1 mutant. Consistent with previous studies, wild type PKS5 protein significantly reduced PM H ATPase activity in vesicles isolated from the pks5 1 mutant and had no effect on the PM H ATPase activity of vesicles isolated from Col 0 plants . Recombinant PKS5 6 protein had no effect on PM H ATPase activity in the vesicles isolated from Col 0 or the pks5 1 and pks5 6 mutants .
When either PKS5 3 or PKS5 4 protein was added to vesicles isolated from Col 0 or the pks5 1 mutant, PM H ATPase activity was reduced; however, sb431542 this effect was much more dramatic in pks5 1 compared with Col 0 . When used as controls, boiled recombinant PKS recombinant proteins did not have any effect on PM H ATPase activity. These results support the conclusion that PKS5 kinase activity is negatively correlated with PM H ATPase activity. J3 Functions Upstream of PKS5 in the Regulation of PM H ATPase Activity To determine whether PKS5 genetically interacts with J3, we crossed j3 1 to pks5 1, pks5 3, or pks5 4 to generate j3 1 pks5 1, j3 1 pks5 3, and j3 1 pks5 4 double mutants. T DNA insertions in pks5 1 and j3 1 were confirmed using gene specific primers, and the pks5 3 and pks5 4 mutations were confirmed using derived cleaved amplified polymorphic sequences primer based PCR followed by sequencing of the mutations.
To assay PM H ATPase activity, plasma membrane enriched vesicles were isolated from Col 0 and double mutant plants treated with or without 250 mM NaCl. As shown in Figure 8, the PM H ATPase activity of the salt treated j3 1 pks5 1 double mutant was similar to the activity of the pks5 1 single mutant, and the activities of both were higher than the activity in Col 0 after salt treatment. These results indicate that, genetically, J3 functions Fostamatinib upstream of PKS5. Furthermore, PM H ATPase activity in both the j3 1 pks5 3 and j3 1 pks5 4 double mutants was similar to the activity of their respective pks5 parent and lower than that of the j3 1 parent . These results demonstrate that J3 regulates PM H ATPase activity by mediating PKS5 kinase activity.