Abstract
Plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a crucial enzyme that exists on the tonoplast to maintain pH homeostasis across the vacuolar membrane. This enzyme generates proton gradient between cytosol and vacuolar lumen by hydrolysis of a metabolic byproduct, pyrophosphate (PPi). The regulation of V-PPase at protein level has drawn attentions of many workers for decades, but its mechanism is still unclear. In this work, we show that AVP1, the V-PPase from Arabidopsis thaliana, is a target protein for regulatory 14-3-3 proteins at the vacuolar membrane, and all twelve 14-3-3 isoforms were analyzed for their association with AVP1. In the presence of 14-3-3nu, -mu, -omicron, and -iota, both enzymatic activities and its associated proton pumping of AVP1 were increased. Among these 14-3-3 proteins, 14-3-3mu shows the highest stimulation on coupling efficiency. Furthermore, 14-3-3nu, -mu, -omicron, and -iota exerted protection of AVP1 against the inhibition of suicidal substrate PPi at high concentration. Moreover, the thermal profile revealed the presence of 14-3-3omicron improves the structural stability of AVP1 against high temperature deterioration. Additionally, the 14-3-3 proteins mitigate the inhibition of Na+ to AVP1. Besides, the binding sites/motifs of AVP1 were identified for each 14-3-3 protein. Taken together, a working model was proposed to elucidate the association of 14-3-3 proteins with AVP1 for stimulation of its enzymatic activity.