Regulation of vacuolar V-ATPase activity and Dynamic control of vacuolar functions

Y.Anraku等人。1980年在酿酒酵母的液泡膜上发现了一种新的ATPase，它是液泡的主要质子泵。该酶是一种至少由13个亚基组成的超大分子复合体。其生理功能被认为参与了液泡功能的动态调控，特别是细胞内钙离子和质子浓度的调节。本课题从细胞生理学的角度对V-ATPase活性和空泡动力学的调控进行了全面的研究，主要结果如下：1)应用一种新建立的全液泡膜片钳方法。我们测定了液泡膜上依赖于ATP的内向电流为100pA，质子/ATP比为3.5。这为该酶在生理条件下作为质子泵的作用提供了直接证据。2)确定了新基因VMA14和VMA15的结构和功能。这些产物对V-ATPase亚单位在液泡膜上的组装具有调节作用。3)分离到一个新基因VIS1，并对其结构进行了测定。该产物(214个氨基酸残基)是嵌入在液泡中的膜蛋白。它的功能与钙敏感生长的调控系统有关，它与UBP信号网络的交叉作用有关。

Y. Anraku et al. found a novel ATPase on the vacuolar membrane of Saccharomyces cerevisiae in 1980 It functions as a major proton pump of the vacuole. The enzyme is a supra-macromolecular complex consisting of at least 13 subunits. Its physiological functions are proposed to participate in dynamic controls of vacuolar functions, particularly the regulation of intracellular calcium ion and proton concentrations. In this project, we intended to study a comprehensive view on the regulation of the V-ATPase activity and vacuolar dynamics in cell physiology.The results are : 1) Applying a newly established method of whole vacuolar patch clamp. we determined an ATP-dependent inward current of 100 pA across the vacuolar membrane with a proton/ATP ratio of 3.5. This provides direct evidence that the enzyme acts as a proton pump under physiological conditions 2) The structure and function of novel genes, VMA14 and VMA15 were determined. The products have a regulatory role for the assembly of subunits of the V-ATPase on the vacuolar membrane. 3) A novel gene, VIS1 was isolated and its structure was determined. The product (214 amino acid residues) is a membrane protein enbedded in the vacuole. Its function relates to a regulatory system for Ca^<2+>-sensitive growth with a cross interaction of the UBP signaling network.