TARGETING OF 2 ARABIDOPSIS H-ATPASE ISOFORMS TO THE PLASMA-MEMBRANE()

More than 11 different P-type H+-ATPases have been identified in Arabi dopsis by DNA cloning. The subcellular localization for individual mem bers 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 an d AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epit ope tags to their C-terminal ends and expressed them in transgenic pla nts. Both tagged isoforms localized to the plasma membrane, as indicat ed 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 plasm a membrane localization. Two epitope tags were evaluated: c-myc, a sho rt, 11-amino acid sequence, and beta-glucuronidase (GUS), a 68-kD prot ein. The c-myc tag is recommended for its sensitivity and specific imm unodetection. CUS worked well as an epitope tag when transgenes were e xpressed at relatively high levels (e.g. with AHA2-CUS944); however, e vidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H+-ATPase complex. Nevertheless, the apparent abilit y 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, solu ble proteins to the plasma membrane.