Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase

Abstract

Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.

Plant proton pumps

“…Furthermore, this model concurs with earlier models derived from immunochemical analysis and computer algorithms predicting that the C‐terminal segment of plant H + ‐PPases is exposed to the cytoplasm [76,77,79,80,87]. However, Van et al have presented an alternative topological model for plant H + ‐PPases using the TopPred II algorithm that predicts the C‐terminal segment exposed to the vacuolar lumen [88,89].…”

“…2) [80,96]. The transmembrane domain 5 (TM5) is highly conserved among plant H + ‐PPases and has a conspicuous lower degree of hydrophobicity that suggest it could be involved in proton translocation [89]. Alanine scanning mutagenesis approach was used to identify critical amino acid residues along TM5 domain of mung bean H + ‐PPase [89].…”

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

“…ϩ -PPases Impaired in Proton Translocation-A previous report demonstrated that Arg-242 in VrH ϩ -PPase mutated to Ala provoked the impairment in both PP i hydrolysis and proton translocating activities (19). In the present study the mutant in which Arg-169 of CtH ϩ -PPase was replaced with the lysine residue possessed similar PP i hydrolysis properties to the cysteine-less mutant.…”

Maize ZmVPP5 is a truncated Vacuole H+‐PPase that confers hypersensitivity to salt stress

“…The VPP family proteins play an important role in Pi balance and in the establishment of a proton motive force across tonoplasts (Ferjani et al ; Li et al ). Previous studies of V‐PPase have mainly focused on the conservative sites and conservative domain structures that are necessary for enzyme activity (Kim et al ; Nakanishi et al ; Van et al ; Asaoka et al ). According to the X‐ray crystal structure, the inside of the VPP ion channels is composed of TM5, TM6, TM11, TM12, TM15 and TM16, the combination of which form the catalytic domain containing the motifs for Mg‐PPi binding, PPi hydrolysis and energy conversion (Leigh et al ; Nakanishi et al ).…”