Properties of polyamine transport proteins and their genes

细胞内多胺含量受多胺生物合成及其转运的双重调控。我们最近在大肠杆菌中获得并鉴定了三个多胺转运基因克隆。其中两个分别是亚精胺优先摄取系统和腐胺特异性摄取系统。亚精胺优先摄取系统由四种蛋白组成：外质底物结合蛋白（pod）、膜相关ATP结合蛋白（potA）和两种跨膜蛋白（pob和C）。腐胺摄取系统还包括四种蛋白质：与亚精胺优先摄取系统相似的蛋白质。通过定点诱变获得的突变蛋白的活性测定和细胞定位发现，ATP结合域和ATP酶活性的活性中心位于potA蛋白的nh_2 -末端和potA与potB、C跨膜蛋白的cooh -末端。通过x射线晶体分析和突变蛋白的活性测定，确定了pod蛋白的亚精胺结合位点。结果发现，Glu36和Glu171对亚精胺的伯胺识别起重要作用，而Asp257对亚精胺的仲胺识别起重要作用。第三个运输系统是腐胺的排泄系统。这是由具有腐胺鸟氨酸反转运蛋白活性的potE蛋白催化的。PotE蛋白由439个氨基酸组成。在potE蛋白的亲水区存在16种酸性氨基酸。通过把它们变成中性氨基酸，我们寻找蛋白质的催化位点。研究发现，Glu207对该活性至关重要。我们还发现RNase III参与了potE基因表达的增强。

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes in Escherichia coli.Two of them were spermidine-preferential and putrescine-specific uptake systems, respectively. Spermidine-preferential uptake system consisted of four kinds of proteins : periplasmic substrate-binding protein (potD) , membrane-associated ATP binding protein (potA) and two transmembrane proteins (potB and C). Putrescine uptake system also consisted of four kinds of proteins : those similar to proteins of the spermidine-preferential uptake system.From activity measurement and cellular localization of mutated proteins obtained by site-directed mutagenesis, it was found that ATP binding domain and active center of ATPase activity were located in the NH_2-terminal of potA protein and COOH-terminal of the protein stimulated association between potA and potB,C transmembrane proteins. Spermidine binding site of potD protein was also determined by X-ray crystal analysis of the purified protein and activity measurement of mutated proteins. It was found that Glu36 and Glu171 were important for the recognition of primary amines and Asp257 was important for the recognition of secondary amine of spermidine.The third transport system was the excretion system of putrescine. This was catalyzed by potE protein, which has putrescine-ornithine antiporter activity. PotE protein consisted of 439 amino acids. There are 16 acidic amino acids existed in the hydrophilic region of potE protein. By changing those to neutral amino acids, we looked for the catalytic site of the protein. It was found that Glu207 was essential for the activity. We also found that RNase III was involved in the enhancement of the expression of potE gene.