Characteristics of polyamine transport and regulation of its gene expression

1. PotF蛋白是大肠杆菌腐胺转运系统的周质底物结合蛋白。我们已经确定了PotF蛋白质的晶体结构在复杂的基板在2.3-_分辨率。PotF分子的尺寸为54 × 42 × 30 - 1，由两个相似的球状结构域组成。腐胺通过12个氢键和36个货车德瓦尔斯相互作用紧密结合在PotF的两个结构域之间的深裂缝中。PotF结构与PotD结构的比较提供了对两种蛋白质之间特异性差异的洞察。PotF结构与突变分析相结合，揭示了腐胺结合的关键残基（Trp-37，Ser-85，Glu-185，Trp-244，Asp-247，和Asp-278）以及水分子对腐胺降解的重要性.使用用该基因转化的酵母细胞检查由YLL 028 w编码的膜蛋白的性质。转化的细胞对聚乙烯产生了抗性， 关于我们 胺毒性，抗性被液泡H^+-ATP酶抑制剂巴弗洛霉素A_1克服。虽然转化细胞的精胺摄取活性几乎与野生型细胞相同，但转化细胞的液泡膜囊泡的摄取活性高于野生型细胞。转化细胞对MGBG（甲基乙二醛双（鸟苷酰腙））和百草枯产生抗性。而Ni^2+和Co^2+不表达，表明YLL 028 w编码的蛋白是一种多胺特异性转运蛋白，表明YLL 028 w编码的膜蛋白（TPO 1）是一种液泡膜上的多胺转运蛋白.利用由potA、-B、-C和-D基因组成的亚精胺摄取操纵子，研究了在存在积累的多胺的情况下，对大肠杆菌亚精胺摄取的抑制。转录的potABCD操纵子被抑制的PotD，亚精胺结合蛋白通常发现在周质中，和PotD的抑制作用增加亚精胺。当模板DNA与PotD的摩尔比约为1：500时，在8 mM亚精胺存在下，PotD的转录抑制率为50%，PotD与potA基因ATG起始密码子上游-258 ~-209和下游+66 ~+135的区域结合。PotD与下游位点的结合受到亚精胺的刺激。PotD在大肠杆菌DH 5 α中的过表达抑制了亚精胺的摄取、PotABCD mRNA的合成以及融合在含有PotD结合位点的potA基因下游的lacZ报告基因的表达。在过表达PotD的细胞中，大量的PotD以前体形式存在于原生质球中。使用突变的PotD蛋白确定了PotD中参与其与potABCD操纵子相互作用的氨基酸残基。PotD的Thr-35和Ser-85在这种相互作用中起重要作用。少

1. PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Eschenchia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-_ resolution. The PotF molecule has dimensions of 54 x 42 x 30 _ and consists of two similar globular domains. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.2. Properties of a membrane protein encoded by YLL028w were examined using yeast cells transformed with the gene. The transformed cells became resistant to poly … More amine toxicity, and the resistance was overcome by bafilomycin A_1, an inhibitor of vacuolar H^+-ATPase. Although spermine uptake activity of the transformed cells was almost the same as that of wild type cells, the uptake activity of vacuolar membrane vesicles from the transformed cells was higher than that from wild type cells. The transformed cells became resistant to MGBG (methylglyoxal bis(guanylhydrazone)) and paraquat. but not Ni^<2+> and Co^<2+>, suggesting that the protein encoded by YLL028w is a transport protein specific for polyamines, The results indicate that a membrane protein encoded by YLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane.3. Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1 : 500 of template DNA : PotD.It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibitedthe uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. Less