Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
EMBO Rep. 2010 Jun;11(6):431-7. doi: 10.1038/embor.2010.51. Epub 2010 May 7.
The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2-C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole-cell patch-clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non-malignant human tissues.
前列腺是一种高度特化的哺乳动物器官，能够产生和释放大量柠檬酸。然而，柠檬酸释放的机制尚不清楚。在这里，我们展示了先前显示具有这种机制的人正常前列腺上皮 PNT2-C2 细胞中柠檬酸转运蛋白的分子克隆结果。通过使用 cDNA 末端快速扩增聚合酶链反应，我们确定前列腺载体是线粒体转运蛋白 SLC25A1 的同工型，其具有不同的第一外显子。我们通过在人胚肾细胞中表达该克隆并进行放射性示踪实验和全细胞膜片钳记录来证实该克隆的功能。通过使用靶向序列不同部分的短干扰 RNA，我们证实克隆的蛋白是负责柠檬酸释放的主要前列腺转运体。我们还制备了特异性抗体，并将克隆的转运蛋白定位于细胞的质膜上。使用相同的抗体，我们已经表明该克隆的转运蛋白在非恶性的人类组织中表达。
