阿拉伯芥RACK1A是否會與14-3-3蛋白有分子上的交互作用？

Abstract

Receptor for Activated C-Kinase 1 (RACK1)是一個分子量為36 kDa的鷹架蛋白，普遍存在於動物和植物中，RACK1的蛋白質結構是由七個平板螺旋 (seven-bladed propeller) 所組成，其中包含七個色胺基酸和天冬胺酸 (tryptophan-aspartic acid-domain；WD40) 的重複區域，而這些重複的區域 (domain) 也是與其他蛋白發生交互作用的地方。在阿拉伯芥中，有三個基因可以轉譯出RACK1蛋白，分別命名為RACK1A (At1g18080), RACK1B (At1g48630) 和RACK1C (At3g18130)。阿拉伯芥中的RACK1蛋白會參與在賀爾蒙反應和植物生長發育的過程中，且會與40S核醣體發生交互作用。目前，RACK1被視為一多功能性的鷹架蛋白，在不同的訊息傳遞途徑中扮演重要的角色。此外，RACK1在離層酸 (abscisic acid; ABA) 反應中扮演一個負向調控的角色，而rack1a突變株對於水分逆境具有抗性。然而，RACK1A究竟是與哪些蛋白質發生交互作用，共同參與或調控這些訊息傳遞的過程，都是尚未清楚的部分。前人研究以14-3-3抗體進行免疫沉澱 (immunoprecipitation) 的實驗，發現到RACK1A的存在。於是，我們想要再進一步去確認14-3-3蛋白與RACK1A蛋白之間的交互作用。由酵母菌雙雜合系統和雙分子螢光互補實驗得知，14-3-3蛋白與RACK1A蛋白之間沒有直接的交互作用。此外，觀察阿拉伯芥RACK1A在原生質體細胞內主要分布在細胞質，C端某絲胺酸定點突變導致RACK1A分布集中在核中。本研究並利用RACK1A大量表現植株，試著將RACK1A及其結合蛋白利用免疫沉澱方式純化出來。

Receptor for Activated C-Kinase 1 (RACK1) is a 36-kDa scaffold protein, which contains seven Trp-Asp 40 (WD40) repeats. RACK1 is highly conserved protein in both animals and plants. The WD40 repeats are involved in protein-protein interactions. There are three RACK1 genes in Arabidopsis thaliana, namely RACK1A (At1g18080), RACK1B (At1g48630) and RACK1C (At3g18130). RACK1 is involved in multiple hormone responses, developmental processes, and associated with 40S ribosomes in Arabidopsis. Notably, RACK1 is regarded as a versatile scaffold protein which serves as a nexus for multiple signal transduction pathways. Moreover, RACK1 plays a negative regulator under abscisic acid (ABA) responses, and rack1a mutants are more resistant to water stress. However, the specific interactions between RACK1 and its binding partners are still unclear. Previous immunopreciptation studies revealed that RACK1A may interact with 14-3-3. Here, we used yeast two-hybrid and bimolecular fluorescence complementation to study the interactions between these two proteins. Our results indicated that RACK1A did not interact with 14-3-3 omega directly. Furthermore, we observed that the RACK1A protein is localized to the cytoplasm in Arabidopsis protoplasts. Point mutation of a serine residue located in the C-terminue resulted in subcellular localization change. We use RACK1A overexpression lines to the possibility to identify RACK1A interacting proteins by immunoprecipitation.