植物硼转运蛋白作用机制的结构和功能研究
硼在植物生长和发育中具有重要作用。 BOR蛋白在硼的主动摄取和分布以及细胞内硼浓度的调节中起关键作用。 但是，它们的作用机理仍然研究不足。 BOR蛋白是SLC4家族转运蛋白的成员，因此是经过充分研究的哺乳动物转运蛋白（包括人类阴离子交换剂1（hAE1））的同源蛋白。 本文，我们基于已知的引起hAE1突变的疾病（S466R，A500R）和ii）在酵母BOR蛋白ScBOR1p中鉴定的功能丧失突变（D311A），产生了拟南芥BOR1（AtBOR1）突变体。 尽管S466R和A500R的表达均低于野生型 AtBOR1和D311A，但突变体在酵母中表达并定位于质膜。 D311A，S466R和A500R突变导致酵母bor1p敲除菌株中硼外流活性的丧失。
含有这三个个体突变的拟南芥植物在低硼条件下在土壤和平板上均表现出明显降低的生长表型。 这些数据证实了D311在蛋白质功能中的重要作用，并表明与引起疾病的hAE1突变等效的突变在AtBOR1中具有重要作用。
我们还从稻（Oryza sativa）OsBOR3获得了BOR蛋白的低分辨率冷冻EM结构。 用于获得该结构的构建体缺少30个C端氨基酸，但未经修饰。 这种结构证实了先前观察到的相关蛋白质的门和核心结构域组织，并且强烈暗示了向内构象。
Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are members of the SLC4 family of transporters and thus homologues of well studied mammalian transporters including the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) mutants based i) on known disease causing mutations of hAE1 (S466R, A500R) and ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The mutants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of boron efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes both in soil and on plates under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3. The construct used to obtain this structure lacks the 30 C-terminal amino acids but is otherwise unmodified. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.
https://www.biorxiv.org/content/10.1101/2020.12.15.422910v1.full.pdf
文章原创，转载请注明来源。
温馨提示：
为方便PaperRSS粉丝们科研、就业等话题交流。我们根据10多个专业方向（植物、医学、药学、人工智能、化学、物理、财经管理、体育等），特建立了30个国内外博士交流群。群成员来源欧美、日韩、新加坡、清华北大、中科院等全球名校。