LAUSR

We provide cryo-EM structures of Drosophila INDY in diverse functional states at near-atomic resolution. Our structures reveal its conformational cycle, sodium-independent H+/citrate co-transport, and phospholipid effects on stability. These findings provide molecular insights into the enigmatic transport mechanism of INDY. Drosophila I’m Not Dead Yet (INDY) functions as a transporter for citrate, a key metabolite in the citric acid cycle, across the plasma membrane. Partial deficiency of INDY extends lifespan, akin to the effects of caloric restriction. In this work, we use cryo-electron microscopy to determine structures of INDY in the presence and absence of citrate and in complex with the well-known inhibitor 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) at resolutions ranging from 2.7 to 3.6 Å. Together with functional data obtained in vitro, the INDY structures reveal the H+/citrate co-transport mechanism, in which aromatic residue F119 serves as a one-gate element. They also provide insight into how protein–lipid interactions at the dimerization interface affect the stability and function of the transporter, and how DIDS disrupts the transport cycle.