1999 Fiscal Year Final Research Report Summary
Molecular biological studies on active ion transporters present in Acetabularia acetabulum
| Project/Area Number |
09640780
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物生理
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| Research Institution | Okayama Prefectural University (1997, 1999)
University of Tsukuba (1998) |
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Principal Investigator |
IKEDA Mikiko Department of Nutritional Science, Faculty of Health & Welfare Science, Okayama Prefectural University Professor, 保健福祉学部, 教授 (20112154)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Acetabularia / Cl^--translocating ATPase / V-ATPase / V-PPase / ABC transporter / cDNA cloning / heterologous expression |
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| Research Abstract |
Molecular biological studies were done for active ion transporters present in Acetabularia acetabulum which had been biochemically investigated by us, namely 1) Cl^--translocating ATPase, 2) two proton pumps (V-ATPase and V-PPase) in vacuolar membrane and 3) ATP-binding cassette type ATPase (ABC transporter). The results obtained for the respective transporter are as following.
1) Cl^--ATPase : Among the three subunits, a (54 kDa), b (50 kDa) and c (40 kDa) the primary structures of the a and b subunits were elucidated by molecular cloning and they showed the highest similarity to the F-type ATPase, α and β subunits, respectively. As for the b subunit, exchangeability with the β subunit of Escherichia coli F_1-ATPase was studied using E.coli F_1-β deficient strain. The polypeptide of the b subunit was incorporated into the membrane fraction, but no oxidative phosphorylation was observed. By analysis of chimeric proteins of the Cl^--b and F_1-β subunit, a region between 96 and 161 amino
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acid was supposed to play important roles in the function.
2) V-ATPase and V-PPase : Two different cDNAs were isolated for the A and B subunits of Acetabularia V-ATPase, respectively and six different cDNAs for the proteolipid subunit. Three of six proteolipid subunits were transformed in Saccharomyces cerevisiae vma3-deficient strain lacking the proteolipid subunit and quinacrine uptake in acidic organelles was observed by fluorescent microscopy. Data preliminarily supported functional complementation of the vma3-deficient strain with the proteolipid subunits of Acetabularia V-ATPase.
One cDNA coding for V-PPase was isolated and sequenced. The primary structure showed 46% identity to R.rubrum and Chara PPase. Acetabularia V-PPase was most effectively expressed in S.cerevisiae BJ5459 strain after recombination of the gene with pYES2 as a yeast expression vector. The expressed PPase was partially purified by hydrophobic and anion-exchange chromatographies and its characteristics as PPase were similar to native Acetabularia V-PPase. As preliminary results, membrane vesicle prepared from the transformant showed H^+- pumping activity.
3) ABC transporter : A full length cDNA coding for the CysA protein of sulfate permease was isolated from Acetabularia. Acetabularia CysA protein consisits of 357 amino acids with a molecular weight of 40,679. The primary structure is 48.7% identical to Anacystis nidulans CysA protein. Less
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