Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds
Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds
复制标题
DOI：
10.1105/tpc.106.045997
复制
发表时间：
2007-02-01
期刊：
影响因子：
11.6
通讯作者：
Hara-Nishimura, Ikuko
中科院分区：
文献类型：
--
作者：
Fuji, Kentaro;Shimada, Tomoo;Hara-Nishimura, Ikuko
拟南芥的两个基因已被证明在种子贮藏蛋白的液泡分选中起作用：一个空泡分选受体VSR1/ATELP1和一个逆转录成分MAIGO1(MAG1)/VPS29。在这里，我们展示了一种有效而简单的方法来分离拟南芥的空泡分离突变体。该方法基于这项研究中的两个发现。首先，VSR1通过识别空泡靶向信号，作为β-伴球蛋白的分选受体发挥作用。其次，当绿色荧光蛋白(GFP)与信号融合(GFP-CT24)在vsr1、mag1/vps29和野生型种子中表达时，vsr1和mag1/vps29都能产生强荧光的种子，而野生型不能，这表明液泡分离的缺陷通过分泌GFP-CT24来提供荧光种子。我们对表达GFP-CT24的转化种子进行了诱变。从300万个M2代种子中，我们获得了>；100荧光种子，并命名为绿色荧光种子突变体。我们报告了10个GFS突变，所有这些突变都导致了储存蛋白的错误分类。我们将gfs1定位到VSR1，gfs2定位到KAM2/GRV2，gfs10定位到编码一个新的膜蛋白基因At4g35870，其余的定位到不同的基因座。这种方法应该为研究贮藏蛋白质液泡分选的复杂分子机制提供有价值的见解。
Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From; 3,000,000 lines of M2 seeds, we obtained > 100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.