V-ATPases are highly regulated proton pumps that acidify organelles. The V-ATPase a-subunit is a two-domain protein containing a C-terminal transmembrane domain responsible for proton transport and an N-terminal cytosolic domain (aNT) that is a regulatory hub, integrating environmental inputs to regulate assembly, localization, and V-ATPase activity. The yeast encodes only two organelle-specific a-isoforms, Stv1 in the Golgi and Vph1 in the vacuole. On the basis of recent structures, we designed chimeric yeast aNTs in which the globular proximal and distal ends are exchanged. The Vph1 proximal-Stv1 distal (VPSD) aNT chimera binds to the glucose-responsive RAVE assembly factor in vitro but exhibits little binding to PI(3,5)P. The Stv1 proximal-Vph1 distal (SPVD) aNT lacks RAVE binding but binds more tightly to phosphoinositides than Vph1 or Stv1. When attached to the Vph1 C-terminal domain in vivo, both chimeras complement growth defects of a mutant, but only the SPVD chimera exhibits wild-type V-ATPase activity. Cells containing the SPVD chimera adapt more slowly to a poor carbon source than wild-type cells but grow more rapidly than wild-type cells after a shift to alkaline pH. This is the first example of a "redesigned" V-ATPase with altered regulatory properties and adaptation to specific stresses.