Wang Feng, Jing Yanping, Wang Zhen, Mao Tonglin, Samaj Jozef, Yuan Ming, Ren Haiyun
State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, China.
J Integr Plant Biol. 2009 Feb;51(2):113-21. doi: 10.1111/j.1744-7909.2008.00781.x.
Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profilin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive localizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.
肌动蛋白结合蛋白（Profilin）是一种对肌动蛋白细胞骨架动力学具有复杂影响的蛋白质。拟南芥中有五种肌动蛋白结合蛋白亚型。然而，这些亚型在功能上是否存在差异仍是一个悬而未决的问题。在本研究中，将拟南芥的两种肌动蛋白结合蛋白亚型PRF1和PRF2与绿色荧光蛋白（GFP）标签融合，并在大肠杆菌和拟南芥中表达，以便比较它们在体外的生化特性以及在体内的细胞分布。生化分析表明，GFP-PRF1和GFP-PRF2的融合蛋白与聚-L-脯氨酸和G-肌动蛋白的结合表现出显著差异。与GFP-PRF2相比，GFP-PRF1对聚-L-脯氨酸和G-肌动蛋白都具有更高的亲和力。对稳定转基因拟南芥品系活细胞的观察表明，35S::GFP-PRF1形成丝状网络，而35S::GFP-PRF2形成多边形网格。用拉特罗毒素A处理及随后的恢复实验结果表明，GFP-PRF1的丝状排列可能与肌动蛋白丝有关。然而，GFP-PRF2定位于类似于内质网的多边形网格中。我们的结果提供了证据，表明拟南芥肌动蛋白结合蛋白亚型PRF1和PRF2对聚-L-脯氨酸和G-肌动蛋白具有不同的生化亲和力，并在活细胞中表现出独特的定位。这些数据表明PRF1和PRF2是功能不同的亚型。
