Quantification, Organ-Specific Accumulation and Intracellular Localization of Type II H+-Pyrophosphatase in Arabidopsis thaliana

Abstract

Most plants have two types of H(+)-translocating inorganic pyrophosphatases (H(+)-PPases), I and II, which differ in primary sequence and K(+) dependence of enzyme function. Arabidopsis thaliana has three genes for H(+)-PPases: one for type I and two for type II. The type I H(+)-PPase requires K(+) for maximal enzyme activity and functions together with H(+)-ATPase in vacuolar membranes. The physiological role of the type II enzyme, which does not require K(+), is not clear. We focused on the type II enzymes (AtVHP2;1 and AtVHP2;2) of A. thaliana. Total amounts of AtVHP2s were quantified immunochemically using a specific antibody and determined to be 22 and 12 ng mg(-1) of total protein in the microsomal fractions of suspension-cultured cells and young roots, respectively, and the values are approximately 0.1 and 0.2%, respectively, of the vacuolar H(+)-PPase. In plants, AtVHP2s were detected immunochemically in all tissues except mature leaves, and were abundant in roots and flowers. The intracellular localization of AtVHP2s in suspension cells was determined by sucrose density gradient centrifugation and immunoblotting. Comparison with a number of marker proteins revealed localization in the Golgi apparatus and the trans-Golgi network. These results suggest that the type II H(+)-PPase functions as a proton pump in the Golgi and related vesicles in young tissues, although its content is very low compared with the type I enzyme.

Keep an Eye on PPi: The Vacuolar-Type H+-Pyrophosphatase Regulates Postgerminative Development inArabidopsis

“…A comparative biochemical characterization between AVP1/FUGU5 and AVP2 (VHP2;1; a type II H + -PPase), after heterologous expression in yeast, revealed that AVP2 is also competent in both PPi hydrolysis and H + -translocation (Drozdowicz et al, 2000), therefore suggesting a role of both H + -PPase types in PPi homeostasis, yet this has to be demonstrated directly in plants (Rea and Sanders, 1987). In our recent study, the total protein amount of the type II enzymes was quantified to be <2.0 ng/mg of protein in the microsomal fraction (Segami et al, 2010). This amount is <0.2% of that of the type I enzyme.…”

“…Arabidopsis has three genes for H + -PPases: a single gene for the type I enzyme, At VHP1;1 (also called AVP1); and two genes for the type II enzyme, At VHP2;1 and At VHP2;2 (Drozdowicz and Rea, 2001;Segami et al, 2010). A comparative biochemical characterization between AVP1/FUGU5 and AVP2 (VHP2;1; a type II H + -PPase), after heterologous expression in yeast, revealed that AVP2 is also competent in both PPi hydrolysis and H + -translocation (Drozdowicz et al, 2000), therefore suggesting a role of both H + -PPase types in PPi homeostasis, yet this has to be demonstrated directly in plants (Rea and Sanders, 1987).…”

The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole inArabidopsis

“…Earlier studies have shown that both enzymes can also be detected in other compartments of the endomembrane system, including the ER, Golgi, and endosomes, and, at least in certain cell types or species, even at the plasma membrane (PM; Herman et al, 1994;Ratajczak et al, 1999;Dettmer et al, 2006;Paez-Valencia et al, 2011). Both enzymes are present in multiple isoforms (Sze et al, 2002;Segami et al, 2010), and their localization has mostly been determined using polyclonal antibodies that did not allow differentiation between individual isoforms . We have shown previously that subcellular distribution of the Arabidopsis V-ATPase is determined by the presence of the respective isoforms of the membrane-integral subunit VHA-a (Dettmer et al, 2006), and here we tracked fully functional fluorescent fusion proteins of VHA-a3 on their way to the tonoplast of the LV in Arabidopsis root tip cells.…”

Genetic Manipulation of a “Vacuolar” H+-PPase: From Salt Tolerance to Yield Enhancement under Phosphorus-Deficient Soils

“…Type II H + -PPases localize and function in the Golgi apparatus, and their total amount in tissues is very low, less than 0.2% of the type I H + -PPase (Segami et al, 2010).…”