Quantification, organ-specific accumulation and intracellular localization of type II H(+)-pyrophosphatase in Arabidopsis thaliana.
Quantification, organ-specific accumulation and intracellular localization of type II H(+)-pyrophosphatase in Arabidopsis thaliana.
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DOI：
10.1093/pcp/pcq096
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发表时间：
2010-08
影响因子：
4.9
通讯作者：
Shoji Segami;Y. Nakanishi;Masa H. Sato;M. Maeshima
中科院分区：
文献类型：
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作者：
Shoji Segami;Y. Nakanishi;Masa H. Sato;M. Maeshima
大多数植物具有两种类型的H（+）-转运无机焦磷酸酶（H（+）-PPases），I和II，它们在一级序列和酶功能的K（+）依赖性上不同。拟南芥具有三个H（+）-PPase基因：一个为I型，两个为II型。I型H（+）-PPase需要K（+）才能达到最大酶活性，并与H（+）-ATPase一起在液泡膜中发挥作用。II型酶不需要K（+），其生理作用尚不清楚。本研究主要对A. thaliana. AtVHP 2s的总量使用特异性抗体进行免疫化学定量，并确定为悬浮培养细胞和幼根的微粒体组分中的总蛋白分别为22和12 ng mg（-1），值分别约为空泡H（+）-PPase的0.1%和0.2%。在植物中，AtVHP 2s在除成熟叶外的所有组织中被免疫化学检测到，并且在根和花中丰富。用蔗糖密度梯度离心和免疫印迹法测定AtVHP 2s在悬浮细胞中的细胞内定位。与一些标记蛋白质的比较显示定位在高尔基体和trans-Golgi网络。这些结果表明，II型H（+）-PPase的功能作为一个质子泵在高尔基体和相关的囊泡在年轻的组织，虽然它的含量是非常低的相比，I型酶。
Most plants have two types of H(+)-translocating inorganic pyrophosphatases (H(+)-PPases), I and II, which differ in primary sequence and K(+) dependence of enzyme function. Arabidopsis thaliana has three genes for H(+)-PPases: one for type I and two for type II. The type I H(+)-PPase requires K(+) for maximal enzyme activity and functions together with H(+)-ATPase in vacuolar membranes. The physiological role of the type II enzyme, which does not require K(+), is not clear. We focused on the type II enzymes (AtVHP2;1 and AtVHP2;2) of A. thaliana. Total amounts of AtVHP2s were quantified immunochemically using a specific antibody and determined to be 22 and 12 ng mg(-1) of total protein in the microsomal fractions of suspension-cultured cells and young roots, respectively, and the values are approximately 0.1 and 0.2%, respectively, of the vacuolar H(+)-PPase. In plants, AtVHP2s were detected immunochemically in all tissues except mature leaves, and were abundant in roots and flowers. The intracellular localization of AtVHP2s in suspension cells was determined by sucrose density gradient centrifugation and immunoblotting. Comparison with a number of marker proteins revealed localization in the Golgi apparatus and the trans-Golgi network. These results suggest that the type II H(+)-PPase functions as a proton pump in the Golgi and related vesicles in young tissues, although its content is very low compared with the type I enzyme.