| Co-Investigator(Kenkyū-buntansha) |
NISHIMURA Kazuhiro Chiba University, Faculty of Pharmaceutical Sciences, Research Assistant, 薬学部, 教務職員 (60302569)
KASHIWAGI Keiko Chiba University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80169424)
KAKINUMA Yoshimi Chiba University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (80134394)
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| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2000: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1999: ¥9,700,000 (Direct Cost: ¥9,700,000)
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| Research Abstract |
1. The effects of polyamines on the synthesis of various σ subunits of RNA polymerase were studied using Western blot analysis. Synthesis of σ^<28> was stimulated 4.0-fold and that of σ^<38> was stimulated 2.3-fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. Stimulation of σ^<28> synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgamo (SD) sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.
2. We identified a gene (TPO1, YLLO28w) that encodes a polyamine transport protein on the vacuolar membrane in yeast. Because the existence of other genes for a polyamine transport protein on the v
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acuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A_1, an inhibitor of the vacuolar H^+- ATPase. Among the four polyamine transporters, those encocded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles.
3. The PotE protein can catalyze both uptake and excetion of putrescine. The Km values of putrescine for uptake and excretion are 1.8 and 73 μM, respectively. Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity. Amino acids involved in both activities were identified using mutated PotE proteins. It was found that Cys^<62>, Trp^<201>, Trp^<292>, and Tyr^<425> were strongly involved in both activities, and that Tyr^<92>, Cys^<210>, Cys^<285>, and Cys^<286> were moderately involved in the activities. Mutations of Tyr^<78>, Trp^<90>, and Trp^<422> mainly affected uptake activity, and the Km values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE.Mutations of Lys^<301> and Tyr^<308> mainly affected excretion activity (putrescine-ornithine antiporter activitiy), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine. Based on the results of competition experimentes with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented. Less
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