University of Amsterdam, Swammerdam Institute for Life Sciences, Section Plant Physiology, Postbus 94215, 1090 GE Amsterdam, The Netherlands.
Biochem J. 2013 Mar 15;450(3):573-81. doi: 10.1042/BJ20121639.
PA (phosphatidic acid) is a lipid second messenger involved in an array of processes occurring during a plant's life cycle. These include development, metabolism, and both biotic and abiotic stress responses. PA levels increase in response to salt, but little is known about its function in the earliest responses to salt stress. In the present study we have combined an approach to isolate peripheral membrane proteins of Arabidopsis thaliana roots with lipid-affinity purification, to identify putative proteins that interact with PA and are recruited to the membrane in response to salt stress. Of the 42 putative PA-binding proteins identified by MS, a set of eight new candidate PA-binding proteins accumulated at the membrane fraction after 7 min of salt stress. Among these were CHC (clathrin heavy chain) isoforms, ANTH (AP180 N-terminal homology) domain clathrin-assembly proteins, a putative regulator of potassium transport, two ribosomal proteins, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and a PI (phosphatidylinositol) 4-kinase. PA binding and salt-induced membrane recruitment of GAPDH and CHC were confirmed by Western blot analysis of the cellular fractions. In conclusion, the approach of the present study is an effective way to isolate biologically relevant lipid-binding proteins and provides new leads in the study of PA-mediated salt-stress responses in roots.
PA（磷酸脂）是一种脂质第二信使，参与植物生命周期中发生的一系列过程。这些过程包括发育、代谢以及生物和非生物胁迫反应。PA 水平会因盐而增加，但对于其在盐胁迫早期反应中的功能知之甚少。在本研究中，我们结合了一种分离拟南芥根外周膜蛋白的方法与脂质亲和纯化，以鉴定与 PA 相互作用并在盐胁迫下被募集到膜上的假定蛋白。通过 MS 鉴定出的 42 种假定的 PA 结合蛋白中，有一组 8 种新的候选 PA 结合蛋白在盐胁迫 7 分钟后在膜部分积累。其中包括网格蛋白重链同工型、AP180 N 端同源域网格蛋白组装蛋白、一种钾转运调节剂、两种核糖体蛋白、GAPDH（甘油醛 3-磷酸脱氢酶）和一种 PI（磷脂酰肌醇）4-激酶。通过对细胞部分的 Western blot 分析，证实了 GAPDH 和 CHC 的 PA 结合和盐诱导的膜募集。总之，本研究的方法是分离生物学上相关的脂质结合蛋白的有效方法，并为研究根中 PA 介导的盐胁迫反应提供了新的线索。
