PtdIns(3,5)P2 is a low-abundance signaling lipid present at < 0.1 % of total PtdIns lipids in yeasts and mammals. Reduced levels of PtdIns(3,5)P2 contributes to neurodegenerative disorders in humans and vacuolar defects in yeasts. Steady-state levels of PtdIns(3,5)P2 are dependent on both its rate of synthesis and turnover. In yeast, PtdIns(3,5)P2 is produced on the vacuole membrane by phosphorylation of PtdIns(3)P at the 5 position of its inositol ring by the Fab1 lipid kinase. Cells lacking Fab1 make no PtdIns(3,5)P2 and exhibit defects in vacuole morphology and function. The lipid phosphatase Fig4 counteracts Fab1 activity by turnover of PtdIns(3,5)P2 into PtdIns(3)P. Vac14 is a regulatory protein implicated in the synthesis and turnover of PtdIns(3,5)P2. It acts as an adaptor protein that controls both of Fab1 and Fig4 proteins. In addition, Vac14 exists as a multimer that allows for self-interaction. However, multimerization state of Vac14 as well as the domain responsible for self-interaction remained unknown. This study aimed to identify the self-interaction domain to elucidate its role in the assembly of the regulatory complex of PtdIns(3,5)P2. The observations seen in this study suggested that Vac14 self-interacts via multiple conserved motifs in the C-terminus, which are crucial for interaction with Fab1 and Fig4, and the normal morphology of yeast vacuoles.