|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1999 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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 amin
o 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.
上記subunitをコードするcDNA6種をクローニングした(AACEVAPD 1-6)。これらのcDNAを酵母発現ベクターpESC-Leuに連結し,AACEVAPD 2,4及び5についてrecombinantを得た。これらを,酵母V-ATPase,proteolipid subunit欠損株(vma3-deficient strain)に導入し,transformantを得た。培養後,酸性小器官へのキナクリンの取り込みを蛍光顕微鏡で観察した。それぞれのtransformantで蛍光がみられ,カサノリV-PPase,proteo-lipid subunitの発現並びに機能のcomplementationが実証された。
生体膜を介する溶質の選択的な移動を媒介するATP-binding cassette(ABC)superfamilyのcDNAクローニングの結果,sulfate permeaseのCysA subunitの全長をコードするcDNAを得た。357アミノ酸をコードし,分子量40,679の蛋白質であり,シアノバクテリアCysA proteinに48.7%のidentityを示すものであった。即ち,バクテリアタイプの構造を有することが明らかになった。 Less