Structure, Function and Evolution of the Vacuolar H+ ATPASE

液泡H+ - atp酶在真核细胞中普遍存在，在植物液泡溶质积累中起核心作用。像fof1 - atp酶一样，它对钒酸盐不敏感，也没有磷酸酶催化中间体。我们和其他实验室已经证明，液泡H+- atp酶是一个大的多聚酶，400-500 kDA，包含三个主要亚基和多达五个次要亚基。目前已知70kda、60kda和16kda亚基的主要结构。70和60 kDa的kDa亚基包含许多高度保守的区域，被认为分别参与F1 β和α亚基的催化和调节。PI希望直接使用化学探针和定点诱变的组合来表征70和60 kDa亚基的功能域。将在大肠杆菌和胡萝卜中进行定点诱变研究。他已经在大肠杆菌中获得了70 kDa亚基的过表达。他计划用反义DNA转化70 kDa cDNA的5'-未翻译区，在胡萝卜中产生空泡型H+- atp酶无突变体。位点定向诱变将通过重新转化无效突变体来进行。系统发育比较将确定液泡H+ atp酶是否与其他古细菌H+- atp酶有关，除了它最近的亲戚Sulfolobus acidocalarius。最后，描述了旨在表征v - atp酶的表面特征和四级结构的电子显微镜研究。

The vacuolar H+ -ATPase is ubiquitous in eukaryotic cells and plays a central role in vacuolar solute accumulation in plants. Like FoF1-ATPases it is insensitive to vanadate and does not have a phosphoenzyme catalytic intermediate. We and other laboratories have shown that the vacuolar H+-ATPase is a large, multimeric enzyme, 400-500 kDA, containing three major subunits an up to five minor ones. The primary structures of the 70 kDa, 60 kDa, and 16 kDa subunits are now known. The 70 and 60 kDa kDa subunits contain numerous highly conserved regions thought to be involved in catalysis and regulation in the F1 beta and alpha subunits, respectively. The PI wishes to characterize the functional domains of the 70 and 60 kDa subunits directly using a combination of chemical probes and site-directed mutagenesis. Site-directed mutagenesis studies will be carried out both in E. coli and in carrot. He has already obtained overexpression of the 70 kDa subunit in E. coli. He plans to generate vacuolar H+-ATPase null mutants in carrot by transforming with antisense DNA to the 5'-untranslated region of 70 kDa cDNA. Site-directed mutagenesis will be carried out by retransforming the null mutants. Phylogenetic comparisons will determine whether or not the vacuolar H+ATPase is related to other archaebacterial H+-ATPases besides that of Sulfolobus acidocaldarius, its closest relative. Finally, electron microscope studies aimed at characterizing the surface features and quaternary structure of the V-ATPase are described.