Marobbio Carlo M T, Giannuzzi Giulia, Paradies Eleonora, Pierri Ciro L, Palmieri Ferdinando
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Bari, Italy.
J Biol Chem. 2008 Oct 17;283(42):28445-53. doi: 10.1074/jbc.M804637200. Epub 2008 Aug 5.
In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.
在酿酒酵母中，在线粒体中产生的α-异丙基苹果酸（α-IPM）必须输出到胞质溶胶中，亮氨酸生物合成需要它在胞质溶胶中发挥作用。重组和重构的线粒体草酰乙酸载体（Oac1p）除了能有效转运其已知底物草酰乙酸、硫酸盐和丙二酸外，还能有效转运α-IPM，这与其他二羧酸和三羧酸转运蛋白以及先前提出的α-IPM转运蛋白不同。转运对α-IPM的半饱和常数为75±4微摩尔，对β-IPM为0.31±0.04毫摩尔，呈饱和状态，并受到Oac1p底物的抑制。虽然亮氨酸生物合成途径中亮氨酸的直接前体α-酮异己酸不被转运，但它能竞争性抑制Oac1p活性。相比之下，亮氨酸、α-酮异戊酸、缬氨酸和异亮氨酸既不抑制Oac1p活性，也不被Oac1p转运。与Oac1p作为α-IPM转运蛋白的功能一致，缺乏该载体基因的细胞在可发酵碳源上生长时需要亮氨酸才能实现最佳生长。其他线粒体载体基因或LEU4（除Oac1p外，唯一能为胞质溶胶提供α-IPM的其他酶）的单基因缺失未表现出生长缺陷，而双突变体DeltaOAC1DeltaLEU4在无亮氨酸的情况下在可发酵底物上根本无法生长。通过向DeltaOAC1DeltaLEU4细胞添加亮氨酸生物合成的胞质中间产物α-酮异己酸和α-IPM，以及用两种未知的人类线粒体载体SLC25A34和SLC25A35之一对其进行互补，部分恢复了DeltaOAC1DeltaLEU4细胞的生长缺陷。Oac1p对在可发酵碳源上的亮氨酸生物合成很重要，它催化α-IPM的输出，可能是与草酰乙酸进行交换。
