National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 210095, Nanjing, People's Republic of China.
Mol Biol Rep. 2010 Feb;37(2):685-93. doi: 10.1007/s11033-009-9535-1. Epub 2009 Apr 17.
RING-finger proteins with E3 ubiquitin ligase activity play important roles in the regulation of plant growth and development. In this study, a cDNA clone encoding a novel RING-finger protein, designated as GmRFP1, was isolated and characterized from soybean. GmRFP1 was an intronless gene encoding a predicted protein product of 392 amino acid residues with a molecular mass of ~43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. The transcript was observed in all detected organs and was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments. We further expressed and purified both wild type and mutant version of GmRFP1 in E. coli. In vitro assays showed that the purified GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING-finger region abolished the ubiquitination activity. These findings suggest that GmRFP1 is a previously unknown E3 ubiquitin ligase in soybean and that the RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.
具有 E3 泛素连接酶活性的环指蛋白在植物生长发育的调控中发挥着重要作用。本研究从大豆中分离并鉴定了一个编码新型环指蛋白的 cDNA 克隆，命名为 GmRFP1。GmRFP1 是一个无内含子的基因，编码一个预测的 392 个氨基酸残基的蛋白质产物，分子量约为 43 kDa。该蛋白含有一个 RING-H2 基序和一个 N 端跨膜结构域。该转录本在所有检测到的器官中均有表达，并受脱落酸（ABA）和盐胁迫诱导上调，但受冷和干旱处理下调。我们进一步在大肠杆菌中表达和纯化了野生型和突变型的 GmRFP1。体外实验表明，纯化的 GmRFP1 诱导多泛素链的形成，而 RING 指区域的突变则使泛素化活性丧失。这些发现表明，GmRFP1 是大豆中一种以前未知的 E3 泛素连接酶，其 RING 结构域是其活性所必需的。它可能通过泛素-蛋白酶体途径介导靶蛋白的泛素化和降解，在 ABA 信号转导和应激反应中发挥未被认识的作用。
