Saku Miyamoto

Excretion of putrescine from Escherichia coli was assessed by measuring its uptake into inside-out membrane vesicles. The vesicles were prepared from wild-type E. coli or E. coli transformed with plasmids containing one of the three polyamine transport systems. The results indicate that excretion of putrescine is catalyzed by the putrescine transport protein, encoded by the potE gene located at 16 min on the E. coli chromosome. Loading of ornithine (or lysine) inside the vesicles was essential for the uptake of putrescine, indicating that the protein exchanges putrescine and ornithine (or lysine) by an antiport mechanism. The Km and Vmax values for the putrescine uptake by inside-out membrane vesicles were 73 microM and 0.82 nmol/min per mg of protein, respectively. The antiport protein (potE protein) also catalyzed putrescine-putrescine and ornithine-ornithine exchange. The transport activity was not disturbed by inhibitors of energy production such as KCN and carbonyl cyanide m-chlorophenylhydrazone. When intact E. coli was used instead of the inside-out membrane vesicles, excretion of putrescine was also catalyzed by the antiport protein in the presence of ornithine in the medium.

Here we show a novel pathway of transcriptional regulation of a DNA-binding transcription factor by coupled interaction and modification (e.g., acetylation) through the DNA-binding domain (DBD). The oncogenic regulator SET was isolated by affinity purification of factors interacting with the DBD of the cardiovascular transcription factor KLF5. SET negatively regulated KLF5 DNA binding, transactivation, and cell-proliferative activities. Down-regulation of the negative regulator SET was seen in response to KLF5-mediated gene activation. The coactivator/acetylase p300, on the other hand, interacted with and acetylated KLF5 DBD, and activated its transcription. Interestingly, SET inhibited KLF5 acetylation, and a nonacetylated mutant of KLF5 showed reduced transcriptional activation and cell growth complementary to the actions of SET. These findings suggest a new pathway for regulation of a DNA-binding transcription factor on the DBD through interaction and coupled acetylation by two opposing regulatory factors of a coactivator/acetylase and a negative cofactor harboring activity to inhibit acetylation.

The nucleotide sequence of the operon for the putrescine transport system that maps at 19 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding potF, -G, -H, and -I proteins. The potF protein (M(r) = 38,000) was inferred to be a putrescine-specific binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. The potG protein (M(r) = 45,000) had consensus amino acid sequences for the nucleotide-binding site. The potH (M(r) = 35,000) and potI (M(r) = 31,000) proteins consisted of six putative transmembrane-spanning segments linked by hydrophilic segments of variable length as shown by hydropathy profiles. The spermidine-putrescine transport system, which is mainly involved in spermidine transport, consisted of potA, -B, -C, and -D proteins (Furuchi, T., Kashiwagi, K., Kobayashi, H., and Igarashi, K. (1991) J. Biol. Chem. 266, 20928-20933). The homologies of the corresponding two proteins between those two systems, F and D, G and A, H and B, and I and C, were 35, 42, 37, and 36%, respectively. The initiation point of the transcription of the operon for the putrescine transport system was determined by primer extension and S1 nuclease mapping. Transcription started from the T residue located either 149 or 150 nucleotides upstream from the initiator AUG codon of potF protein mRNA. By making several subclones and a mutant lacking the potF gene, we showed that the expression of all four proteins was necessary for maximal putrescine transport activity. These results indicate that the putrescine transport system can also be defined as a bacterial periplasmic transport system.