Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

Abstract

In plants, the proton pump‐ATPase (H(+)‐ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)‐ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)‐ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C‐terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild‐type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2‐ to 3‐fold increase of H(+)‐pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)‐ATPase improve the coupling between H(+)‐pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild‐type PMA2, indicating a relationship between H(+)‐ATPase activity and perturbations of the secretory pathway.

Summary

“…Glucose dependent H ϩ extrusion from yeast cells was measured essentially as reported in Morsomme et al (1996). The cell suspension was stabilized at pH 5.5 by titration with NaOH and HCl before starting the assay.…”

“…The acidification of the external medium by yeast cells can be used as a measure for the activity of H ϩ -ATPase in the plasma membrane (Baunsgaard et al, 1996;Morsomme et al, 1996). When glucose was added to the medium as a carbon source the cells responded by extruding H ϩ (Figure 1b).…”

“…Single point mutations in the autoinhibitory domain can produce a constitutively active H ϩ -ATPase (Baunsgaard et al, 1996;Morsomme et al, 1996), and the C-terminal regulatory domain has recently been found to be associated with the 14-3-3 protein (Jahn et al, 1997;Oecking et al, 1997), of which there are several isoforms. 14-3-3 proteins have been implicated in the co-ordination of signal transduction pathways (Aitken, 1996) and in the regulation of an increasing number of enzymes (Sehnke and Ferl, 1996).…”

Blue light activates the plasma membrane H+-ATPase by phosphorylation of the C-terminus in stomatal guard cells

“…The H ϩ -ATPase activity is thought to be regulated by an autoinhibitory domain in the C-terminal region of the enzyme. Removal of a 7-10 kDa fragment from the C-terminal end of H ϩ -ATPase generates a high-activity state of H ϩ -ATPase with a higher V max , a lower K m for ATP, a changed pH dependence with higher activity at physiological pH and an increased coupling of H ϩ transport with ATP hydrolysis (Palmgren et al, 1990;Morsomme et al, 1996;Sze et al, 1999). Very similar results were obtained by in vivo treatment of intact tissue with the fungal toxin fusicoccin (FC), an activator of the H ϩ -ATPase, and it is suggested that FC induces a displacement of the C-terminal autoinhibitory domain of H ϩ -ATPase from its catalytic site (Palmgren, 1991;Johansson et al, 1993;Rasi-Caldogno et al, 1993).…”

“…ATP hydrolysis is sufficient to account for the observed H ϩ pumping, since the enzyme is assumed to have a stoichiometry of 2H ϩ / ATP (Spanswick, 1981) or H ϩ /ATP (Briskin and ReynoldsNiesman, 1991). It is also possible that BL concomitantly improves the coupling between H ϩ pumping and ATP hydrolysis (Baunsgaard et al, 1996;Morsomme et al, 1996). (A) BL-dependent H ϩ pumping in GCPs was measured by the decrease of pH in the medium.…”