The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates
The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates
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DOI：
10.1042/bj20060409
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发表时间：
2006-07-01
影响因子：
4.1
通讯作者：
Aronson, Peter S.
中科院分区：
文献类型：
--
作者：
Knauf, Felix;Mohebbi, Nilufar;Aronson, Peter S.
在黑腹果蝇中发现了一种名为Indy（意为“我还没死”）的长寿基因，它与编码二羧酸钠协同转运蛋白的哺乳动物基因相似。在非洲爪蟾卵母细胞中的功能研究表明，INDY介导二羧酸和柠檬酸跨质膜的通量，但由INDY介导的具体运输机制尚未确定。为了测试是否INDY功能作为阴离子交换剂，我们检查了是否基板外排刺激可运输的基板添加到外部介质。通过向外部培养基中添加琥珀酸盐，大大加速了[C-14]柠檬酸盐从表达INDY的卵母细胞中的流出，表明柠檬酸盐-琥珀酸盐交换。琥珀酸盐刺激的[C-14]柠檬酸盐流出对DIDS（4，4 '-二异硫氰基-2，2'-二磺酸芪）的抑制敏感，如先前对INDY介导的琥珀酸盐摄取所证明的。INDY介导的[C-14]柠檬酸盐外排也受到外部柠檬酸盐和草酰乙酸盐的刺激，表明柠檬酸盐-柠檬酸盐和柠檬酸盐-草酰乙酸盐交换。类似地，来自INDY表达卵母细胞的[C-14]琥珀酸酯的ef-flux被外部柠檬酸盐、α-酮戊二酸盐和富马酸盐刺激，分别表明琥珀酸酯-柠檬酸盐、琥珀酸酯-α-酮戊二酸盐和琥珀酸酯-富马酸盐交换。相反，当表达INDY的非洲爪蟾卵母细胞加载琥珀酸和柠檬酸盐时，[C-14]琥珀酸摄取被显著刺激，证实了琥珀酸-琥珀酸和琥珀酸-柠檬酸交换。内部阴离子与外部柠檬酸盐的交换显著pH值。依赖性，与柠檬酸盐与质子共转运的概念一致。阴离子交换不依赖于钠。我们的结论是，INDY功能作为一个交换器的二羧酸和三羧酸Krebs循环中间体。在长寿的Indy突变体中，降低INDY活性的效果可能是以有利于寿命延长的方式改变能量代谢。
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 [C-14]citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate-succinate exchange. The succinate-stimulated [C-14]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 [C-14]citrate was also stimulated by external citrate and oxaloacetate, indicating citrate-citrate and citrate-oxaloacetate exchange. Similarly, ef-flux of [C-14]succinate from INDY-expressing oocytes was stimulated by external citrate, a-oxoglutarate and fumarate, indicating succinate-citrate, succinate-a-oxoglutarate and succinate-fumarate exchange respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [C-14]succinate uptake was markedly stimulated, confirming succinate-succinate and succinate-citrate exchange. Exchange of internal anion for external citrate was markedly pH.-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.