Isolation and characterization of plant plasma membrane P-type H+-ATPase dimers.

Plant H⁺-ATPases are activated through tightly regulated hexameric assembly, relieving autoinhibition via displacement of a regulatory domain during the oligomerization process. Sub-hexameric assembly states also exist but their role in activity regulation remains elusive. To characterize sub-hexameric plant H⁺-ATPases, we isolated homo-oligomers of the H⁺-ATPases AHA2 and determined their sizes with size exclusion chromatography multi-angle light scattering. AHA2 predominantly presented as a monomer but also assembled into stable dimers and larger complexes. Activity assays on solubilized and liposome-reconstituted AHA2 monomers and dimers revealed that dimerization did not affect enzymatic activity, suggesting that AHA2 dimerization does not represent a separate regulatory pathway. Instead, AHA2 dimers may serve as reservoirs for rapid hexamer assembly, facilitating the dynamic regulation of protein activity.