Maeda S, Omata T
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan.
J Biol Chem. 1997 Jan 31;272(5):3036-41. doi: 10.1074/jbc.272.5.3036.
Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 microM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.
在蓝藻聚球藻属PCC 7942菌株中，硝酸盐主动运输所需的四个基因（nrtABCD）中，nrtBCD编码参与亚硝酸盐以及硝酸盐运输的ATP结合盒转运体的膜成分，而nrtA编码一种45 kDa的细胞质膜蛋白，其生化功能尚不清楚。对nrtA缺失突变体的表征表明，这种45 kDa的蛋白对于硝酸盐/亚硝酸盐转运体的功能至关重要。在大肠杆菌细胞中作为组氨酸标签可溶性蛋白表达的、缺少N端81个氨基酸的截短NrtA蛋白，被证明能以高亲和力（Kd = 0.3 microM）结合硝酸盐和亚硝酸盐。使用针对这种45 kDa蛋白的抗体进行的免疫印迹分析揭示了该蛋白的一个48 kDa前体，它在经球霉素处理的蓝藻细胞中积累，球霉素是一种特异性抑制脂蛋白前体信号肽切割的抗生素。这些发现表明，nrtA基因产物是一种结合硝酸盐和亚硝酸盐的脂蛋白。假定的蓝藻底物结合蛋白的N端序列表明，ATP结合盒转运体的底物结合蛋白的脂蛋白修饰在蓝藻中很常见。
