Department of Biochemistry, University College of Science, Osmania University, Hyderabad 500007, India.
Fungal Genet Biol. 2011 Nov;48(11):1020-6. doi: 10.1016/j.fgb.2011.07.006. Epub 2011 Aug 5.
Our previous in silico studies identified a high-affinity nickel transporter, TNC, from the metal transportome of Neurospora crassa. A knockout mutant of the tnc gene in N. crassa failed to transport nickel, showed phenotypic growth defects and diminished urease activity under physiological levels of nickel. Transport assays conducted in wild type and knockout mutant strains showed that TNC transports nickel with high affinity but exhibits selectivity for other transition metal ions like cobalt. Heterologous complementation of Schizosaccharomyces pombe nickel uptake mutant by TNC further substantiates its nickel transport function. Transcriptional analysis of the nickel transporter encoding gene, tnc in N. crassa by qRT-PCR showed its constitutive expression in various phases of its life cycle. However, levels of the corresponding protein TNC were down-regulated only by increasing the nickel, but not cobalt concentration in the media. Immunolocalisation data suggested that TNC is distributed in the plasma membrane of N. crassa. Thus, the present study establishes TNC as a functional plasma membrane nickel transporter necessary for physiological acquisition of nickel in the multicellular fungi N. crassa.
我们之前的计算机研究从粗糙脉孢菌的金属转运组中鉴定出一种高亲和力镍转运蛋白 TNC。粗糙脉孢菌 tnc 基因的敲除突变体无法转运镍，在生理水平的镍下表现出表型生长缺陷和脲酶活性降低。在野生型和敲除突变体菌株中进行的转运实验表明，TNC 以高亲和力转运镍，但对其他过渡金属离子（如钴）表现出选择性。TNC 通过异源互补酿酒酵母镍摄取突变体进一步证实了其镍转运功能。通过 qRT-PCR 对粗糙脉孢菌镍转运蛋白编码基因 tnc 的转录分析表明，其在生命周期的各个阶段均持续表达。然而，只有增加培养基中的镍而不是钴浓度，才能下调相应蛋白 TNC 的水平。免疫定位数据表明，TNC 分布在粗糙脉孢菌的质膜上。因此，本研究确立了 TNC 作为一种功能性质膜镍转运蛋白，对于多细胞真菌粗糙脉孢菌中镍的生理获取是必需的。
