Regulation of polyamine contents in cells

1. PotD蛋白是一种质周结合蛋白，是多胺转运系统的主要受体。在2.5 *分辨率下对pod与亚精胺配合物的晶体结构进行了解析。PotD蛋白由两个具有交替- α - β拓扑结构域组成。多胺结合位点位于两个结构域之间界面的中心裂缝中。通过位点定向诱变制备的突变pod蛋白，并通过测量这些突变pod蛋白的多胺结合活性，研究了pod上亚精胺的结合位点。结果表明，pod蛋白的Trp-34、Thr-35、Clu-36、Tyr-37、Ser-83、Tyr-85、Asp-168、Glu-171、Trp-229、Trp-255、Asp-257、Tyr-293和Gln-327参与了亚精胺与pod蛋白的结合，其中Glu-171、Trp-255和Asp-257参与亚精胺与pod蛋白结合的作用比其他氨基酸更强。研究了多胺转运蛋白PotE的结构和功能。PotE对腐胺的吸收依赖于膜电位。相反，在内外膜囊泡中研究的腐胺-鸟氨酸反转运蛋白活性不依赖于膜电位。腐胺摄取和腐胺-鸟氨酸反转运蛋白活性的Km值分别为1.8和73 muM。因此，PotE不仅可以作为腐胺-鸟氨酸反转运蛋白排泄腐胺，还可以作为腐胺摄取蛋白。不同PotE融合蛋白对碱性磷酸酶和β -半乳糖苷酶的激活表明，PotE的NH_2和COOH端均位于细胞质中。利用突变的PotE蛋白研究了腐胺摄取和排泄的活性。研究发现，谷氨酸207是PotE蛋白摄取和排泄腐胺所必需的，而谷氨酸77和谷氨酸433也参与了这两种活动。这三种谷氨酸位于PotE的细胞质侧，这三种残基的功能不能被其他氨基酸取代。少

1. PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system. The crystal structure of PotD in complex with spermidine has been solved at 2.5-* resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. Spermidine binding sites on PotD were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. It was found that Trp-34, Thr-35, Clu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine, and that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids … More listed above.2. The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential. The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 muM,respectively. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH_2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE and the function of these three residues could not be replaced by other amino acids. Less