Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.
J Biol Chem. 2010 Jun 4;285(23):17359-70. doi: 10.1074/jbc.M109.097188. Epub 2010 Apr 6.
Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP(+) and GSH/GSSG ratios in the cytosol of DeltaYHM2 cells as well as an increase in the NADPH/NADP(+) ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the DeltaYHM2 strain and more so by the DeltaYHM2DeltaZWF1 strain upon H(2)O(2) exposure, implying that Yhm2p has an antioxidant function.
线粒体载体是一类穿梭于线粒体膜内外的转运蛋白，可以转运代谢物、核苷酸和辅酶。目前仅有少数 35 种酿酒酵母线粒体载体的功能尚未被揭示。在本研究中，我们对酿酒酵母线粒体载体 Yhm2p 进行了功能定义和特征描述。在酿酒酵母中过表达 YHM2 基因，纯化其产物并将其重构成脂质体。其转运特性、动力学参数和靶向线粒体的特性表明，Yhm2p 是柠檬酸和草酰琥珀酸的线粒体转运蛋白。重组 Yhm2p 也能转运一定程度的草酰乙酸、琥珀酸和富马酸，但几乎不能转运苹果酸和异柠檬酸。Yhm2p 只催化反交换转运，该转运是饱和的，可被巯基阻断剂抑制，但不能被 1,2,3-苯三甲酸（柠檬酸/苹果酸载体的强抑制剂）抑制。Yhm2p 的生理作用是增加细胞质中的 NADPH 还原能力（用于合成和抗氧化反应），可能作为线粒体和细胞质之间柠檬酸-草酰琥珀酸 NADPH 氧化还原穿梭的关键组成部分。该蛋白功能基于以下观察结果：与野生型细胞相比，ΔYHM2 细胞的细胞质中 NADPH/NADP(+) 和 GSH/GSSG 比值降低，线粒体中 NADPH/NADP(+) 比值增加。我们的建议还得到了ΔYHM2 菌株在过氧化氢暴露时显示的生长缺陷以及ΔYHM2ΔZWF1 菌株生长缺陷更严重的支持，这意味着 Yhm2p 具有抗氧化功能。
