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中的一种周质底物结合蛋白。我们测定了钾F蛋白与底物形成的配合物的晶体结构，分辨率为2.3。钾氟分子的尺寸为54×42×30，由两个相似的球状结构域组成。腐胺通过12个氢键和36个van der Waals相互作用紧密结合在PotF的两个结构域之间的深裂缝中。通过对PotF和PotD结构的比较，可以深入了解这两种蛋白质在特异性上的差异。结合突变分析，PotF结构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)以及水分子对腐胺识别的重要性。将YLL028w编码的膜蛋白转化酵母细胞，对其性质进行了检测。转化的细胞对聚…产生了抗药性。这种抗性可被液泡H~+-ATPase抑制剂巴非霉素A_1克服。尽管转化细胞对精胺的摄取活性与野生型细胞几乎相同，但转化细胞对液泡膜囊泡的摄取活性高于野生型细胞。转化的细胞对MGBG和百草枯产生抗药性。而不是Ni^&lt；2+&gt；和Co^&lt；2+&gt；，这表明YLL028w编码的蛋白是多胺专一性的转运蛋白，结果表明YLL028w(TPO1)编码的一个膜蛋白是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，研究了在聚集多胺存在的情况下对大肠杆菌摄取亚精胺的抑制作用。PotD是一种通常存在于周质中的亚精胺结合蛋白，它抑制了PotABCD操纵子的转录，而亚精胺则增强了PotD的抑制作用。在8 mM亚精胺存在下，模板DNA：PotD的摩尔比约为1：500，对转录有50%的抑制作用。发现PotD结合在POTA基因ATG起始密码子上游的-258-209个核苷酸和+66-+135个核苷酸的下游。亚精胺可刺激PotD与下游结合。在大肠杆菌DH5α中过表达PotD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。在高表达PotD的细胞中，球体中存在大量的PotD前体。用突变的PotD蛋白测定了PotD中参与其与PotABCD操纵子相互作用的氨基酸残基。发现PotD的Thr-35和Ser-85在这种相互作用中起重要作用。较少

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