Guarino Francesca, Specchia Valeria, Zapparoli Giada, Messina Angela, Aiello Rita, Bozzetti Maria Pia, De Pinto Vito
Department of Chemical Sciences, University of Catania, Catania, Italy.
Biochem Biophys Res Commun. 2006 Aug 4;346(3):665-70. doi: 10.1016/j.bbrc.2006.05.172. Epub 2006 Jun 6.
Mitochondrial porins or VDACs (voltage-dependent anion-selective channels) are transmembrane pore-forming proteins. In eukaryotic genomes multiple genes coding for VDAC homologues have been discovered, but their function remains unknown. In Drosophila melanogaster three additional genes homologous to the gene porin have been found. In a previous report we have expressed in vitro Porin 2 (gene GC17137) and we have found that the reconstituted protein shows pore-forming activity but it is cation-selective and poorly dependent from voltage. In this work we have characterized the expression pattern of Porin 2. Amplification upon germinal and somatic or stage specific mRNA showed that the highest transcription level of Porin 2 is in testis. Western blot analysis performed with antibodies raised against the recombinant Porin 2 confirmed a high level of expression in the fly spermatozoa. Immuno-histochemical studies indicate that Porin 2 is selectively present in spermatozoa tail, where the mitochondria are located, but not in spermatocytes. A lethal mutant of D. melanogaster carrying a P-element in the first intron of the porin (Porin 1) gene hinders the expression of both Porin 1 and 2. Our results suggest that Porin 2 is truly expressed and that it is required for functional germinal tissues.
线粒体孔蛋白或电压依赖性阴离子选择性通道(VDACs)是形成跨膜孔的蛋白质。在真核生物基因组中,已发现多个编码VDAC同源物的基因,但其功能仍不清楚。在黑腹果蝇中,已发现另外三个与孔蛋白基因同源的基因。在之前的一份报告中,我们在体外表达了孔蛋白2(基因GC17137),并且发现重组蛋白具有成孔活性,但它具有阳离子选择性,且对电压的依赖性较差。在这项工作中,我们对孔蛋白2的表达模式进行了表征。对生殖细胞、体细胞或阶段特异性mRNA的扩增显示,孔蛋白2的最高转录水平在睾丸中。用针对重组孔蛋白2产生的抗体进行的蛋白质印迹分析证实,其在果蝇精子中的表达水平很高。免疫组织化学研究表明,孔蛋白2选择性地存在于精子尾部(线粒体所在的位置),而不存在于精母细胞中。携带位于孔蛋白(孔蛋白1)基因第一个内含子中的P因子的黑腹果蝇致死突变体阻碍了孔蛋白1和2的表达。我们的结果表明,孔蛋白2确实表达,并且是功能性生殖组织所必需的。