Differential response of vacuolar proton pumps to osmotica.

The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. The higher-plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, the pumps respond differentially to stress in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. No significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts.

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

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.

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.