Fujimoto Masaru, Suda Yasuyuki, Vernhettes Samantha, Nakano Akihiko, Ueda Takashi
Laboratory of Developmental Cell Biology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Present address: Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan.
RIKEN Center for Advanced Photonics, Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan Present address: Department of Molecular Cell Biology, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan.
Plant Cell Physiol. 2015 Feb;56(2):287-98. doi: 10.1093/pcp/pcu195. Epub 2014 Dec 15.
The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.
纤维素微纤丝在植物细胞壁中的定向沉积在多种植物功能中起着关键作用，如细胞生长、器官形成和防御反应。纤维素由嵌入质膜（PM）的纤维素合酶复合体（CSCs）合成，这些复合体包含纤维素合酶（CESAs）。为了精确调节纤维素沉积，质膜上CSCs的丰度和定位应受到严格控制，而这在很大程度上依赖于膜运输系统。然而，CSCs的细胞内运输机制仍知之甚少。在本研究中，我们通过分析拟南芥中表达绿色荧光蛋白标记的CESA3（GFP-CESA3）的PI合成抑制剂的作用，探索了CESA运输中对磷酸肌醇（PIs）的需求。我们发现，转移到无蔗糖条件下会加速质膜定位的GFP-CESA3通过富含网格蛋白的反式高尔基体网络（TGN）重新定位到高尔基体周边。用渥曼青霉素（Wm）（一种磷脂酰肌醇3-激酶（PI3K）和4-激酶（PI4K）的抑制剂）和氧化苯胂（PAO）（一种对PI4K更具特异性的抑制剂）处理，抑制了GFP-CESA3从质膜的内化。相反，用损害PI3K活性的LY294002处理，对GFP-CESA3的隔离没有产生这种抑制作用，但导致GFP-CESA3在高尔基体的环形周边大量积累，从而使GFP-CESA3从质膜上移除。这些结果表明，PIs是CESA3定位和细胞内运输的必需元素，并且PI4K和PI3K是CESA3分泌和/或内吞运输不同步骤所必需的。
