Cloning of the genes for polyamine transport proteins and regulation of its expression

已知多胺摄取在细胞增殖过程中增加，并依赖于能量。我们最近成功地在大肠杆菌中获得了多胺转运基因的三个克隆(pPT104、pPT79和pPT71)。亚精胺的摄取是由pPT104编码的系统催化的，在没有亚精胺的情况下，腐胺的摄取是由pPT104和pPT79编码的系统催化的。PPT79系统对腐胺的摄取不受亚精胺的影响，而pPT104系统对腐胺的摄取受到极大的抑制。这两个系统由四种蛋白质组成：一种周质底物结合蛋白(分别针对pPT104和pPT79的PotD和PotF蛋白)，一种具有核苷酸结合位点的膜相关蛋白(POTA和POTG蛋白)，以及另外两种具有6个跨膜跨膜片段的膜蛋白(针对pPT104的PotB和C蛋白，以及针对pPT79的POTH和I蛋白)。膜小泡和缺乏Fo、F0、F1-ATPase的大肠杆菌突变体对多胺的摄取均依赖于ATP和周质底物结合蛋白。腐胺的排泄是由pPT71催化的。该克隆编码MR46K蛋白，由12个跨膜蛋白片段组成，这些跨膜片段由长度可变的亲水性片段连接。该蛋白是一种介于腐胺和鸟氨酸(或赖氨酸)之间的反向转运蛋白。KCN和间氯苯肼(CCCP)等能量产生抑制剂不干扰其转运活性。

Polyamine uptake is known to increase during cell proliferation and to be energy dependent. We recently succeeded in obtaining three clones of polyamine transport genes (pPT104, pPT79, and pPT71) in E. coli. Spermidine uptake was catalyzed by the system encoded by pPT104, and putrescine uptake was catalyzed by the systems encoded by pPT104 and pPT79 in the absence of spermidine. Putrescine uptake by the pPT79 system was not influenced by spermidine, whereas that by the pPT104 system was greatly inhibited. These two systems consisted of four kinds of proteins: a periplasmic substrate binding protein (potD and potF proteins for pPT104 and pPT79, respectively), a membrane associated protein having the nucleotide binding site (potA and potG proteins) and two other membrane proteins having 6 putative transmembrane spanning segments (potB and C proteins for pPT104, and potH and I proteins for pPT79). The polymine uptake was shown to be both ATP- and periplasmic substrate binding protein-dependent, using membrane vesicles and E. coli mutants lacking Fo, F_0, F_1-ATPase. Excretion of putrescine was catalyzed by pPT71. The clone encoded Mr 46K protein which consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length. The protein was an antiport protein between putrescine and ornithine (or lysine). The transport activity was not disturbed by the inhibitors of energy production such as KCN and carbonylcyanide m-chlorophenylhydrazone (CCCP).