The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates

Abstract

A longevity gene called Indy (for 'I'm not dead yet'), with similarity to mammalian genes encoding sodium-dicarboxylate cotransporters, was identified in Drosophila melanogaster. Functional studies in Xenopus oocytes showed that INDY mediates the flux of dicarboxylates and citrate across the plasma membrane, but the specific transport mechanism mediated by INDY was not identified. To test whether INDY functions as an anion exchanger, we examined whether substrate efflux is stimulated by transportable substrates added to the external medium. Efflux of [14C]citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate-succinate exchange. The succinate-stimulated [14C]citrate efflux was sensitive to inhibition by DIDS (4,4'-di-isothiocyano-2,2'-disulphonic stilbene), as demonstrated previously for INDY-mediated succinate uptake. INDY-mediated efflux of [14C]citrate was also stimulated by external citrate and oxaloacetate, indicating citrate-citrate and citrate-oxaloacetate exchange. Similarly, efflux of [14C]succinate from INDY-expressing oocytes was stimulated by external citrate, alpha-oxoglutarate and fumarate, indicating succinate-citrate, succinate-alpha-oxoglutarate and succinate-fumarate exchange respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [14C]succinate uptake was markedly stimulated, confirming succinate-succinate and succinate-citrate exchange. Exchange of internal anion for external citrate was markedly pH(o)-dependent, consistent with the concept that citrate is co-transported with a proton. Anion exchange was sodium-independent. We conclude that INDY functions as an exchanger of dicarboxylate and tricarboxylate Krebs-cycle intermediates. The effect of decreasing INDY activity, as in the long-lived Indy mutants, may be to alter energy metabolism in a manner that favours lifespan extension.

Figure 1. Effects of external substrates on [¹⁴C]citrate efflux from INDY-expressing Xenopus oocytes

INDY-expressing oocytes and water-injected oocytes not expressing INDY were microinjected with [¹⁴C]citrate and then washed and transferred to various external solutions: NaCl buffer with 1 mM of the indicated substrate, or NaCl buffer with 1 mM of the indicated substrate and 100 μM DIDS. The appearance of radioactivity in the external solution during a 3 min incubation was determined by scintillation spectroscopy. Results are means±S.E.M. *P<0.05. N.S., not statistically significant.

Figure 2. Effects of external substrates on [¹⁴C]succinate efflux from INDY-expressing Xenopus oocytes

INDY-expressing oocytes and water-injected oocytes not expressing INDY were microinjected with [¹⁴C]succinate and then washed and transferred to various external solutions: NaCl buffer with 1 mM of the indicated substrate, or NaCl buffer with 1 mM of the indicated substrate and 100 μM DIDS. The appearance of radioactivity in the external solution during a 3 min incubation was determined by scintillation spectroscopy. Results are means±S.E.M. *P<0.05. N.S., not statistically significant. α-ketoglutarate, α-oxoglutarate.

Figure 3. Effects of internal substrates on [¹⁴C]succinate uptake into INDY-expressing Xenopus oocytes

INDY-expressing oocytes were microinjected with 50 nl of water or a 1 M solution of the indicated substrate. Oocytes were then washed and transferred to NaCl buffer containing [¹⁴C]succinate with or without 100 μM DIDS. After 3 min of incubation, external isotope was removed by washing the oocytes with ice-cold buffer, and radioisotope content was measured by scintillation spectroscopy. Results are means±S.E.M. *P<0.05. N.S., not statistically significant.

Figure 4. Effects of external pH on citrate- and succinate-stimulated [¹⁴C]citrate efflux from INDY-expressing Xenopus oocytes

INDY-expressing oocytes were microinjected with [¹⁴C]citrate and then washed and transferred to various external solutions: NaCl buffer with 1 mM succinate or citrate at pH 6.0, pH 7.5 and pH 9.0. The appearance of radioactivity in the external solution during a 3 min incubation was determined by scintillation spectroscopy. Results are means±S.E.M. *P<0.05. N.S., not statistically significant.