Takeuchi M, Ueda T, Sato K, Abe H, Nagata T, Nakano A
Molecular Membrane Biology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
Plant J. 2000 Aug;23(4):517-25. doi: 10.1046/j.1365-313x.2000.00823.x.
Protein secretion plays an important role in plant cells as it does in animal and yeast cells, but the tools to study molecular events of plant secretion are very limited. We have focused on the Sar1 GTPase, which is essential for the vesicle formation from the endoplasmic reticulum (ER) in yeast, and have previously shown that tobacco and Arabidopsis SAR1 complement yeast sar1 mutants. In this study, we have established a transient expression system of GFP-fusion proteins in tobacco and Arabidopsis cultured cells. By utilizing confocal laser scanning microscopy, we demonstrate that a dominant negative mutant of Arabidopsis Sar1 inhibits the ER-to-Golgi transport of Golgi membrane proteins, AtErd2 and AtRer1B, and locates them to the ER. The same mutant Sar1 also blocks the exit from the ER of a vacuolar storage protein, sporamin. These results not only provide the first evidence that the Sar1 GTPase functions in the ER-to-Golgi transport in plant cells, but also prove that conditional expression of dominant mutants of secretory machinery can be a useful tool in manipulating vesicular trafficking.
蛋白质分泌在植物细胞中与在动物和酵母细胞中一样起着重要作用，但用于研究植物分泌分子事件的工具非常有限。我们聚焦于Sar1 GTP酶，它在酵母中对于从内质网（ER）形成囊泡至关重要，并且我们之前已经表明烟草和拟南芥的SAR1能够互补酵母sar1突变体。在本研究中，我们在烟草和拟南芥培养细胞中建立了绿色荧光蛋白（GFP）融合蛋白的瞬时表达系统。通过利用共聚焦激光扫描显微镜，我们证明拟南芥Sar1的显性负性突变体抑制了高尔基体膜蛋白AtErd2和AtRer1B从内质网到高尔基体的转运，并将它们定位在内质网中。相同的突变体Sar1也阻断了液泡储存蛋白伴刀豆球蛋白从内质网的输出。这些结果不仅首次证明Sar1 GTP酶在植物细胞的内质网到高尔基体转运中发挥作用，而且还证明分泌机制显性突变体的条件性表达可以成为操纵囊泡运输的有用工具。
