Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany AS CR, Sokolovská 6, Olomouc 77200, Czech Republic.
BMC Plant Biol. 2012 Jun 7;12:83. doi: 10.1186/1471-2229-12-83.
RanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized.
Based on sequence similarity, we identified a homologue of the human RanBPM in Arabidopsis thaliana. AtRanBPM protein has highly conserved SPRY, LisH, CTLH and CRA domains. Cell fractionation showed that endogenous AtRanBPM or expressed GFP-AtRanBPM are mainly cytoplasmic proteins with only a minor portion detectable in microsomal fractions. AtRanBPM was identified predominantly in the form of soluble cytoplasmic complexes ~230-500 kDa in size. Immunopurification of AtRanBPM followed by mass spectrometric analysis identified proteins containing LisH and CRA domains; LisH, CRA, RING-U-box domains and a transducin/WD40 repeats in a complex with AtRanBPM. Homologues of identified proteins are known to be components of the C-terminal to the LisH motif (CTLH) complexes in humans and budding yeast. Microscopic analysis of GFP-AtRanBPM in vivo and immunofluorescence localization of endogenous AtRanBPM protein in cultured cells and seedlings of Arabidopsis showed mainly cytoplasmic and nuclear localization. Absence of colocalization with γ-tubulin was consistent with the biochemical data and suggests another than a centrosomal role of the AtRanBPM protein.
We showed that as yet uncharacterized Arabidopsis RanBPM protein physically interacts with LisH-CTLH domain-containing proteins. The newly identified high molecular weight cytoplasmic protein complexes of AtRanBPM showed homology with CTLH types of complexes described in mammals and budding yeast. Although the exact functions of the CTLH complexes in scaffolding of protein degradation, in protein interactions and in signalling from the periphery to the cell centre are not yet fully understood, structural conservation of the complexes across eukaryotes suggests their important biological role.
RanBPM（微管组织中心的Ran 结合蛋白）最初在人类细胞中被报道为中心体相关蛋白。然而，含有高度保守 SPRY、LisH、CTL 和 CRA 结构域的 RanBPM 蛋白目前被认为是一种具有多种细胞功能的支架蛋白。植物 RanBPM 的同源物尚未被描述。
基于序列相似性，我们在拟南芥中鉴定出人类 RanBPM 的同源物。AtRanBPM 蛋白具有高度保守的 SPRY、LisH、CTL 和 CRA 结构域。细胞分级显示，内源性 AtRanBPM 或表达的 GFP-AtRanBPM 主要是细胞质蛋白，只有一小部分可检测到微粒体部分。AtRanBPM 主要以大小约 230-500 kDa 的可溶性细胞质复合物的形式存在。AtRanBPM 的免疫沉淀随后进行质谱分析，鉴定出含有 LisH 和 CRA 结构域的蛋白质；LisH、CRA、RING-U-box 结构域和与 AtRanBPM 形成复合物的转导素/WD40 重复。鉴定出的蛋白质的同源物已知是人类和芽殖酵母中 LisH 基序（CTL）复合物 C 端的组成部分。GFP-AtRanBPM 在体内的显微镜分析和内源性 AtRanBPM 蛋白在培养细胞和拟南芥幼苗中的免疫荧光定位显示主要在细胞质和核定位。与 γ-微管蛋白无共定位与生化数据一致，并表明 AtRanBPM 蛋白的作用不同于中心体。
我们表明，尚未被描述的拟南芥 RanBPM 蛋白与含有 LisH-CTL 结构域的蛋白质发生物理相互作用。新鉴定的 AtRanBPM 的高分子细胞质蛋白复合物与哺乳动物和芽殖酵母中描述的 CTLH 类型的复合物具有同源性。尽管 CTLH 复合物在蛋白质降解支架、蛋白质相互作用以及从细胞边缘向细胞中心的信号转导中的确切功能尚未完全了解，但复合物在真核生物中的结构保守表明其具有重要的生物学作用。
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