Ohnishi Miwa, Yoshida Katsuhisa, Mimura Tetsuro
Department of Biology, Graduate School of Science, Kobe University, Rokkodai 1-1, Nada, Kobe, 657-8501, Japan.
Methods Mol Biol. 2018;1696:107-116. doi: 10.1007/978-1-4939-7411-5_7.
A large number of proteins in the vacuolar membrane (VM; tonoplast), including transporters and receptors, support the various functions of the vacuole. Molecular analysis of membrane proteins is an essential step in understanding how the vacuole operates but so far only a small number of tonoplast proteins have been identified at the molecular level. Accordingly, mutant lines with altered level of tonoplast proteins for characterizing their physiological roles have been developed sparsely. Also, detecting activities of tonoplast proteins remains difficult as it requires a certain degree of enrichment of this organelle fraction. In order to extend our understanding of the vacuole, several groups have turned to proteomic analysis of tonoplast membrane proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high so that its composition can be accurately analyzed with mass spectrometry. In this chapter, we describe a simple method for the isolation of intact vacuoles and subsequent proteome analysis of the VM fraction of cells from Arabidopsis suspension cultures.
液泡膜（VM；液泡膜）中的大量蛋白质，包括转运蛋白和受体，支持着液泡的各种功能。膜蛋白的分子分析是理解液泡如何运作的关键步骤，但到目前为止，只有少数液泡膜蛋白在分子水平上得到鉴定。因此，用于表征其生理作用的液泡膜蛋白水平改变的突变株系开发得很少。此外，检测液泡膜蛋白的活性仍然很困难，因为这需要对该细胞器部分进行一定程度的富集。为了拓展我们对液泡的理解，几个研究小组转向了液泡膜蛋白的蛋白质组学分析。蛋白质组学对任何细胞器进行分析的首要要求是分离出的细胞器纯度要高，以便能够用质谱准确分析其组成。在本章中，我们描述了一种从拟南芥悬浮培养细胞中分离完整液泡并随后对液泡膜部分进行蛋白质组分析的简单方法。
