<p>Cation/H⁺ exchangers (CAXs) mediate vacuolar Ca²⁺ sequestration and are critical for maintaining cytosolic Ca²⁺ homeostasis in plants. Arabidopsis CAX1, a member of the Ca²⁺/Cation Antiporter (CaCA) superfamily, features a modular architecture comprising two pseudosymmetrical domains separated by a cytosolic loop called the acidic motif. CAX1 is also regulated by a cytosolic N-terminal autoinhibitory domain. To define the structural basis of CAX1 activity, we characterized truncated constructs of the N-terminal half of CAX1, comprising a 6-transmembrane (TM) module lacking the autoinhibitory domain (½N-sCAX1), using yeast complementation, structural modeling, and protein interaction studies. The ½N-sCAX1 monomer folded into a stable topology but it failed to interact with itself or with full-length CAX1, or confer transport activity. Functional reconstitution required tethering two ½N-sCAX1 modules via the acidic motif or removal of TM1, which restored partial Ca²⁺ transport in yeast. Protein interaction assays revealed that the autoinhibitory domain contributes to ½N-CAX1 dimerization, while TM1 interferes with complex assembly. Structural models demonstrated that correct alignment of the conserved GNxxE motif across ½N-sCAX1 monomers, either by artificial tethering or potentially by higher order hexameric oligomerization, is essential to reconstruct a functional Ca²⁺-binding pocket. These findings show that CAX1 functionality depends on specific topological constraints and modular interactions that guide formation of CAX1 halves. Our results highlight how architectural features such as TM1 and the autoinhibitory domain regulate transporter assembly and activity, offering insight into CaCA biogenesis and providing a framework for engineering transporters with tailored functional properties.</p>