Abstract
A vacuole is a membrane-enclosed fluid-filled cavity found in the cells of plants and fungi. There are two proton pumps in the tonoplast, vaculoar H+-pyrophosphatases (V-PPase; EC 3.6.1.1) and vaculoar H+-ATPase (V-ATPase; EC 3.6.1.3). They play a main role in catalyzing electrogenic H+-translocation from the cytosol to the vacuolar lumen to generate an inside-acidic and inside-positive membrane potential. V-PPase uses a simple, low-cost substrate pyrophosphate (PPi) as an energy source. Meanwhile, V-PPase contains only a single type of polypeptide of 80 kDa with 14-16 transmembrane domains (TMs) rendering a good model for studying the coupling mechanism between proton translocation and PPi hydrolysis. Among these transmembrane domains, TM 11 is highly conserved in many organisms. In this study, the residues in TM 11 of mung bean V-PPase were mutated by means of alanine-substituted mutagenesis. Mutated genes were over-expressed in Saccharomyces cerevisiae, and the V-PPase-enriched microsomes were prepared. The PPi hydrolysis activities, proton translocation, and coupling efficiency of the mutant V-PPases were then determined. K541A was the only mutant whose enzymatic activity decreased dramatically. It is apparent that the residue Lys-541 is important for the function of V-PPase. In addition, five significant residues (Ser-547, Leu-553, Leu-555, Phe-556 and Ala-558) were identified presumably to be involved in coupling mechanism. Furthermore, the ion effects, thermostability, and proteolytic analysis of these mutants were measured. There are some variations in degree of K+-stimulation for these mutants. Besides, we also found that the thermal stability of V560A and S561A is much higher than wild type. The susceptibilities of several mutant V-PPases to trypsin digestion in the absence of physiological substrate Mg2PPi were different, suggesting the variation in their conformation. The importance of each amino acid residues along TM11 is discussed. A working model of V-PPase is proposed accordingly.