Targeting of Two Arabidopsis H+-ATPase Isoforms to the Plasma Membrane

Abstract

More than 11 different P-type H+-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H+-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1–193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and β-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H+-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H+-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.