College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Department of Plant Cell Biology, Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Science park 904, 1098 XH, Amsterdam, The Netherlands.
J Integr Plant Biol. 2017 Dec;59(12):851-865. doi: 10.1111/jipb.12578. Epub 2017 Oct 18.
The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicle-tethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunit-specific function, and provided data and tools for further characterization of PATL family proteins.
外被体是一种众所周知的复合物，它在融合前将囊泡固定在细胞膜上。单个亚基是否能执行独特的功能在很大程度上是未知的。通过酵母双杂交 (Y2H) 分析，我们发现 EXO70A1 与 Patellin3 (PATL3) 的 GOLD 结构域相互作用。体外和体内实验都支持 EXO70A1 与 PATL3 的直接相互作用。在拟南芥中，PATL3-GFP 主要与 EXO70A1 在细胞膜上共定位，并且 PATL3 的定位对 BFA 和 TryA23 不敏感。值得注意的是，在 exo70a1 突变体中，PATL3 蛋白积累为细胞质内的点状结构，不与几个内质网隔室标记物共定位，并且对 BFA 不敏感。此外，在 exo70e2、PRsec6 或 exo84b 突变体中，PATL3 的定位没有改变。这些数据表明，EXO70A1 而不是其他外被体亚基负责 PATL3 的定位，这与其在分泌/回收囊泡 - tethering /融合中的作用无关。体外实验表明 EXO70A1 和 PATL3 都能结合 PI4P 和 PI(4,5)P。有证据表明，PATL 家族的其他四个成员也与 EXO70A1 结合，并与 PATL3 具有相似的定位模式。这些发现为外被体亚基特异性功能提供了新的见解，并为进一步表征 PATL 家族蛋白提供了数据和工具。
