Perera I Y, Li X, Sze H
Department of Plant Biology, University of Maryland, College Park 20742, USA.
Plant Mol Biol. 1995 Oct;29(2):227-44. doi: 10.1007/BF00043648.
To understand the subcellular roles and the regulation of vacuolar H(+)-ATPases, we have begun to identify the genes encoding the major subunits and to determine their patterns of expression in Arabidopsis thaliana. Two distinct cDNAs (AVA-P1 and AVA-P2) and one genomic sequence (AVA-P3) encoding the 16 kDa subunit have been isolated. The 16 kDa proteolipid is a major component of the membrane integral sector that forms the proton conductance pathway and is required for assembly of the V-ATPase complex. Interestingly, the open reading frame of one full-length cDNA (AVA-P1) and a genomic sequence (AVA-P3) encoded an identical polypeptide of 164 amino acids with a molecular mass of 16,570. The deduced amino acid sequences of the two cDNAs were nearly identical (99%) and hydropathy plots suggested a molecule with four membrane-spanning domains characteristic of V-ATPase proteolipids. The three genes differed mainly in their codon usage and in their 3'-untranslated regions. The coding region of the genomic sequence, AVA-P3, was interrupted by two introns located at the codons for Cys-26 and Arg-121. The presence of additional 16 kDa proteolipid genes was suggested from several polymerase chain reaction (PCR)-amplified fragments that differed from one another in the size of the second intron. PCR 1 had an intron of ca. 800 bp and its identity as AVA-P4, a fourth member of the gene family, was confirmed from sequence analyses of an EST cDNA. The mRNAs of three genes (AVA-P1, AVA-P2 and AVA-P3) were detected in Arabidopsis leaf, root, flower and silique; yet expression of AVA-P1 and AVA-P2 was lower in roots. All three genes were expressed in light- or dark-grown seedlings; however mRNA levels of AVA-P2 were enhanced in etiolated plants. Arabidopsis thaliana, therefore, has at least four distinct genes encoding nearly identical 16 kDa proteolipids, and the enhanced expression of AVA-P2 transcript in etiolated seedlings suggests that an increase in V-ATPase could accompany cell expansion.
为了解液泡H⁺-ATP酶的亚细胞功能及调控机制，我们已开始鉴定编码主要亚基的基因，并确定它们在拟南芥中的表达模式。现已分离出两个不同的cDNA（AVA-P1和AVA-P2）以及一个编码16 kDa亚基的基因组序列（AVA-P3）。16 kDa的蛋白脂质是膜整合区的主要成分，形成质子传导途径，是V-ATP酶复合体组装所必需的。有趣的是，一个全长cDNA（AVA-P1）和一个基因组序列（AVA-P3）的开放阅读框编码了一个由164个氨基酸组成、分子量为16,570的相同多肽。两个cDNA推导的氨基酸序列几乎相同（99%），亲水性图谱表明该分子具有V-ATP酶蛋白脂质特有的四个跨膜结构域。这三个基因主要在密码子使用和3'非翻译区存在差异。基因组序列AVA-P3的编码区被位于Cys-26和Arg-121密码子处的两个内含子打断。从几个聚合酶链反应（PCR）扩增片段推测存在其他16 kDa蛋白脂质基因，这些片段的第二个内含子大小不同。PCR 1有一个约800 bp的内含子，通过对一个EST cDNA的序列分析证实其为该基因家族的第四个成员AVA-P4。在拟南芥的叶、根、花和角果中检测到了三个基因（AVA-P1、AVA-P2和AVA-P3） 的mRNA；然而，AVA-P1和AVA-P2在根中的表达较低。所有三个基因在光照或黑暗培养的幼苗中均有表达；然而，在黄化苗中AVA-P2的mRNA水平有所提高。因此，拟南芥至少有四个不同的基因编码几乎相同的16 kDa蛋白脂质，并且黄化苗中AVA-P2转录本的表达增强表明V-ATP酶的增加可能与细胞扩张有关。
