Dietz K J, Rudloff S, Ageorges A, Eckerskorn C, Fischer K, Arbinger B
Julius-von-Sachs-Institut für Biowissenschaften, Julius-Maximilians-Universität Würzburg, Germany.
Plant J. 1995 Oct;8(4):521-9. doi: 10.1046/j.1365-313x.1995.8040521.x.
A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two-dimensional gels. Amino acid sequences were determined from LysC endoproteinase-peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H(+)-ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26,228 g mol-1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free-flow electrophoresis. Immunoprecipitation of newly synthesized 35S-labelled membrane proteins with anti-TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co-purification of TpP 31 with the 55 kDa subunit of the H(+)-ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H(+)-ATPase.
从二维凝胶中分离出一种表观分子量为31 kDa的液泡膜蛋白（TpP 31）。通过赖氨酸C内蛋白酶肽片段测定氨基酸序列。利用简并寡核苷酸，从大麦叶片cDNA文库中分离出一个1034 bp的相应cDNA克隆。它编码液泡H(+)-ATP酶的E亚基，这是迄今为止在植物中鉴定出的第一个此类亚基。开放阅读框延伸超过681 bp，编码一个由227个氨基酸组成的基因产物，计算分子量为26,228 g mol-1。Northern和Western印迹分析表明，E亚基在所有植物器官中组成性表达，盐胁迫的影响较小。通过自由流动电泳获得的纯化液泡膜组分证实了TpP 31定位于液泡膜。用抗TpP 31对新合成的35S标记膜蛋白进行免疫沉淀，得到另外两条表观分子量约为53和62 kDa的条带。温和溶解后的凝胶过滤显示TpP 31与H(+)-ATP酶的55 kDa亚基共纯化。这两个结果都提供了除序列同源性之外的证据，证明TpP 31是液泡H(+)-ATP酶的结构成分。
