Drosophila INDY and Mammalian INDY: Major Differences in Transport Mechanism and Structural Features despite Mostly Similar Biological Functions

Abstract

INDY (I'm Not Dead Yet) is a plasma membrane transporter for citrate, first identified in Drosophila. Partial deficiency of INDY extends lifespan in this organism in a manner similar to that of caloric restriction. The mammalian counterpart (NaCT/SLC13A5) also transports citrate. In mice, it is the total, not partial, absence of the transporter that leads to a metabolic phenotype similar to that caloric restriction; however, there is evidence for subtle neurological dysfunction. Loss-of-function mutations in SLC13A5 (solute carrier gene family 13, member A5) occur in humans, causing a recessive disease, with severe clinical symptoms manifested by neonatal seizures and marked disruption in neurological development. Though both Drosophila INDY and mammalian INDY transport citrate, the translocation mechanism differs, the former being a dicarboxylate exchanger for the influx of citrate^2- in exchange for other dicarboxylates, and the latter being a Na⁺-coupled uniporter for citrate^2-. Their structures also differ as evident from only ~35% identity in amino acid sequence and from theoretically modeled 3D structures. The varied biological consequences of INDY deficiency across species, with the beneficial effects predominating in lower organisms and detrimental effects overwhelming in higher organisms, are probably reflective of species-specific differences in tissue expression and also in relative contribution of extracellular citrate to metabolic pathways in different tissues.

Keywords: Drosophila INDY; I’m Not Dead Yet; Na+-coupled citrate transporter; SLC13A5; caloric restriction; citrate in metabolism; dicarboxylate exchanger; lifespan; mammalian INDY; transport mechanism.

Figure 1

Schematic representation of functional features of transport via Drosophila INDY and human INDY (SLC13A5/NaCT). (A) Electroneutral transport of citrate (divalent form citrate²⁻) in exchange for dicarboxylates by Drosophila INDY; (B) Electrogenic transport of citrate (divalent form citrate²⁻) coupled to cotransport of 3Na⁺. H⁺ (or Li⁺) can replace at least one or two, but not all three, Na⁺-binding sites. For both Drosophila INDY and human INDY, we postulate that the active transporter is a homodimer; each monomer is represented by a rectangle inserted in the lipid bilayer.

Figure 2

Chemical structures of substrates for Drosophila INDY. At physiological pH, citrate exists as a trivalent anion, whereas all other substrates exist as divalent anions.

Figure 3

Sequence alignment of Drosophila INDY and human INDY (NaCT). Dots below the sequence indicate binding residues for citrate (red) and Na⁺ (gray) based on the cryo-EM structure of human INDY [28]. The degree of residue conservation increases from light blue to dark blue. Red asterisks above the sequence show amino acids in Drosophila INDY that are different from the amino acids constituting the Na⁺ and citrate binding sites in human INDY. The SNT/V motifs are shown in red boxes. The accession numbers for the protein sequences are Q9VVT2 for Drosophila INDY and Q86YT5 for human INDY.

Figure 4

Inward-facing models for Drosophila INDY and human INDY (NaCT). (A) The 3D model for Drosophila INDY, created with AlphaFold, shows an inward-facing conformation dimer. The N-terminal extensions are extensive and are predicted as largely unstructured and located in the cytoplasm. Drosophila INDY is larger than human INDY and shows a C-terminal α-helix located at the extracellular side, which is not evident in human INDY. The N-terminus is predicted to reside on the cytoplasmic side and the C-terminus in the extracellular side; (B) the cryo-EM dimer structure of human INDY in an Inward- facing conformation. Na⁺ ions are shown as purple spheres and citrate as yellow sticks.

Figure 5

Close-up view of proposed binding sites for citrate and α-ketoglutarate in human INDY and Drosophila INDY. Na⁺ ions are shown as purple spheres; citrate (yellow) and α-ketoglutarate (magenta) are shown as sticks.