Perin M S, Fried V A, Stone D K, Xie X S, Südhof T C
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1991 Feb 25;266(6):3877-81.
A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.
最近发现一种116千道尔顿的多肽是从多种来源分离出的液泡质子泵的常见组分。质子泵的116千道尔顿亚基是从牛脑的网格蛋白包被小泡中纯化得到的，并从蛋白水解肽段获得了内部序列。根据这些肽段序列设计的寡核苷酸探针被用于聚合酶链反应，以分离该蛋白的部分牛cDNA克隆。然后利用这些克隆的序列分离出包含全长编码区的大鼠脑cDNA克隆。RNA印迹显示脑中存在一条丰富的3.9千碱基的116千道尔顿亚基的信息，引物延伸分析表明克隆的序列是全长的。大鼠cDNA序列预测会合成一种96267道尔顿的蛋白质。对116千道尔顿亚基推导的氨基酸序列分析表明，它由两个基本结构域组成：一个亲水性的氨基末端，其中带电荷的残基超过30%；一个疏水性的羧基末端，包含至少六个跨膜区域。116千道尔顿质子泵多肽的结构特性与其在将细胞质亚基的ATP水解与泵的膜内成分的质子转运偶联中的假定功能非常吻合。
