Molecular biological studies on active ion transporters present in Acetabularia acetabulum

本文对我们已经研究过的髋臼中存在的离子转运蛋白进行了分子生物学研究，它们是：1）Cl^-转运ATP酶，2）液泡膜上的两个质子泵（V-ATP酶和V-PPase），3）ATP结合盒型ATP酶（ABC转运蛋白）。1）Cl^--ATP酶：在三个亚基a（54 kDa）、B（50 kDa）和c（40 kDa）中，a和B亚基的一级结构通过分子克隆被阐明，它们分别与F-型ATP酶的α和β亚基显示出最高的相似性。对于B亚基，利用E.coliF_1-β缺陷株研究了其与E.coliF_1-ATPase β亚基的交换作用。多肽的B亚基被纳入膜部分，但没有观察到氧化磷酸化。通过对C1 ^-B和F_1-β亚基嵌合蛋白的分析，发现C1 ^-B和F_1-β亚基之间的96 - 161个氨基酸的区域， ...更多信息 2）V-ATPase和V-PPase：分离到了伞藻V-ATPase A和B亚基的两个不同的cDNA，以及蛋白脂亚基的六个不同的cDNA。六个蛋白脂质亚基中的三个转化在酿酒酵母vma 3缺陷株缺乏蛋白脂质亚基和奎纳克林摄取酸性细胞器中观察到的荧光显微镜。初步证实vma 3缺陷株与伞藻V-ATPase的蛋白脂亚基功能互补。其一级结构与红色轮藻和轮藻PPase的同源性为46%。将该基因与酵母表达载体pYES 2重组后，在酿酒酵母BJ 5459中得到了高效表达。表达的PPase通过疏水层析和阴离子交换层析部分纯化，其特性与天然伞藻V-PPase相似。初步结果表明，从伞藻中制备的膜囊泡具有H^+泵活性。3）ABC转运蛋白：从伞藻中分离到一个编码硫酸盐通透酶CysA蛋白的全长cDNA。CysA蛋白由357个氨基酸组成，分子量为40，679。一级结构与Anacystis nidulans CysA蛋白的同源性为48.7%。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