Molecular biological studies on active ion transporters present in Acetabularia acetabulum

我们对髋臼中存在的活性离子转运体进行了分子生物学研究，即1)Cl^-易位ATPase， 2)空泡膜上的两个质子泵（V-ATPase和V-PPase）和3)atp结合盒型ATPase （ABC转运体）。对各自的转运体得到的结果如下。1) Cl^——ATPase：在a （54 kDa）、b （50 kDa）和c (40 kDa) 3个亚基中，通过分子克隆鉴定了a和b亚基的一级结构，它们分别与f型ATPase、α和β亚基具有最高的相似性。在大肠杆菌F_1-β缺陷菌株中，研究了b亚基与大肠杆菌F_1- atp酶β亚基的交换性。b亚基的多肽被纳入膜组分，但未观察到氧化磷酸化。通过对Cl^- b和F_1-β亚基嵌合蛋白的分析，发现在96 ~ 161个氨基酸之间有一个区域，更多的氨基酸在其功能中起重要作用。2) V-ATPase和V-PPase：分别为髋臼V-ATPase的A亚基和B亚基分离到2个不同的cdna，为蛋白脂质亚基分离到6个不同的cdna。6个蛋白脂亚基中的3个在缺乏蛋白脂亚基的酿酒酵母vma3缺陷菌株中转化，荧光显微镜观察到酸性细胞器对醌的摄取。数据初步支持vma3缺陷菌株与髋臼v - atp酶蛋白脂亚基的功能性互补。分离并测序了一个V-PPase编码cDNA。初级结构与红草和Chara PPase的同源性为46%。将该基因与酵母表达载体pYES2重组后，在酿酒酵母BJ5459中最有效地表达了乙酰臼V-PPase。经疏水层析和阴离子交换层析纯化得到的PPase与天然乙酰臼V-PPase相似。初步结果表明，转化制备的膜泡具有H^+泵送活性。3) ABC转运体：从髋臼中分离到硫酸渗透酶CysA蛋白的全长cDNA。Acetabularia CysA蛋白由357个氨基酸组成，分子量为40679。初级结构与Anacystis nidulans CysA蛋白有48.7%的相似性。少

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 … More 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