Complementation in situ of the yeast plasma membrane H⁺‐ATPase gene pmal by an H⁺‐ATPase gene from a heterologous species

Abstract

In plants and fungi, the transport of solutes across the plasma membrane (pm) is driven by a proton pump (H+‐ATPase) that produces an electric potential and a pH gradient. We expressed AHA2, a member of the Arabidopsis thaliana pm H+‐ATPase gene family, in yeast cells in which transcription of the endogenous pm H+ATPase gene (pmal) had been turned off. AHA2 was expressed mainly in intracellular membranes and only supported very slow growth of transformed yeast cells. Removal of the last 92 C‐terminal amino acids from the plant H+‐ATPase produced an enzyme with 2–3‐fold higher specific ATPase activity than the wild‐type plant enzyme. Surprisingly, the truncated H+‐ATPase was now targetted to the yeast pm and fully supported normal yeast growth.

Summary

“…In this study a yeast strain was employed in which the endogenous plasma membrane H ϩ -ATPase PMA1 can be replaced by the plasma membrane H ϩ -ATPase AHA2 from Arabidopis thaliana (Palmgren and Christensen, 1993). On glucose medium, this yeast strain expresses only AHA2 and not PMA1.…”

“…AHA2 is a poor substitute for PMA1, apparently because AHA2 cannot be activated post-translationally in yeast (Palmgren and Christensen, 1993; Figure 1a). The ATPase aha2∆92 is a truncated hyperactive form of AHA2, which lacks the C-terminal regulatory domain and which supports yeast growth to about the same extent as PMA1 (Palmgren and Christensen, 1993;Figure 1a). Fusicoccin was found to stimulate the growth of cells expressing AHA2 (Figure 1a) albeit not to the same level as that of cells expressing aha2∆92.…”

“…The Arabidopsis thaliana plasma membrane H ϩ -ATPase AHA2 and a deletion mutant encoding a protein lacking 92 C-terminal amino acids (aha2∆92) (Palmgren and Christensen, 1993) were expressed in yeast from a multicopy vector under control of the constitutive PMA1 promoter (Villalba et al, 1992).…”

“…In order to identify components necessary for fusicoccininduced activation of the plant plasma membrane H ϩ -ATPase, we have employed a yeast system in which growth is coupled to activation of this enzyme (Palmgren and Christensen, 1993). We found that the yeast responds to fusicoccin by increased growth and activation of H ϩ extrusion, demonstrating that all components required for fusicoccin activation of heterologously expressed plant H ϩ -ATPase are present in yeast.…”

Structure, function and regulation of plasma membrane H+‐ATPase

“…This mechanism of regulation seems also to apply to the plant ATPase, where removal of the carboxyl-terminus at either the protein [22] or gene [23] level activates the enzyme. In addition, a peptide including the region of homology to the yeast inhibitory domain (Fig.…”