Interaction Analyses of Arabidopsis Tubby-Like Proteins with ASK Proteins

阿拉伯芥TULP和ASK蛋白質之交互作用分析

賴嘉萍(Chia-Ping Lai) ； 蕭介夫(Jei-Fu Shaw)

《Botanical Studies》 53卷4期 (2012/10) Pp. 447-458

摘要

本研究利用酵母菌雙雜合系統（yeast two-hybrid system）研究10個阿拉伯芥類肥胖蛋白質（AtTLPs）和不同的阿拉伯芥ASKs間的交互作用情形，結果顯示6個ATTLPs（AtTLP1、2,、3、9、10及11）能夠專一性的和ASK1作用；AtTLP6能和ASK1、ASK2及ASK11交互作用，而AtTLP5、AtTLP7及AtTLP8則和任何的ASK蛋白質無交互作用之情形；利用AtTLP2進行系列剃除（Serial deletion）分析，結果顯示F-box domain及tubby domain均為AtTLP2和ASK1交互作用所需，應用功能性區塊互換（domain swapping）的技術進行分析後，我們推論AtTLP7的N端F-box domain上及AtTLP5的C端tubby domain上的胺基酸自然變異，可能導致AtTLP7及AtTLP5無法和ASK1交互作用；進一步應用定點突變（site-directed mutagenesis）之技術，我們進一步驗證AtTLP7之F-box domain上脯胺酸（proline）突變成白胺酸（leucine）及半胱胺酸（cysteine）突變成絲胺酸（serine）的自然變異，可能為AtTLP7無法和ASK1交互作用的原因。綜合這些發現，我們推論可以專一性和ASK1作用之AtTLP1、2、3、9、10和11可能具有重疊之功能，而AtTLP5、7及AtTLP8不能和任何的ASK作用，推論可能在演化過程喪失原始功能和／或獲得新的功能。

關鍵字

類肥胖基因 ； F-box蛋白質 ； 阿拉伯芥 ； 酵母菌雙雜合系統 ； 蛋白質-蛋白質交互作用 ； ASK

並列摘要

In this study, we have investigated protein-protein interactions between 10 Arabidopsis TUBBY- like proteins (AtTLPs) and various Arabidopsis Skp1-like proteins (ASKs) using the yeast two-hybrid system. The results indicate that six AtTLPs (AtTLP1, 2, 3, 9, 10, 11) are able to specifically interact with ASK1. AtTLP6 is able to interact with ASK1, ASK2, and ASK11, while AtTLP5, AtTLP7, and AtTLP8 did not show any significant interaction with any ASK protein. Serial deletion analyses of AtTLP2 have demonstrated that both F-box domain and tubby domain are required for AtTLP2-ASK1 interaction. Domain swapping suggests that variations at the N-terminus of AtTLP7 and C-terminus of AtTLP5 result in the inability to interact with ASK1. Using site-directed mutagenesis, we further demonstrated that naturally occurring proline-to-leucine and cysteine-to-serine variations in F-box domain might result in the dysfunction of AtTLP7's interaction with ASK1. Taking these observations together, we propose that AtTLP1, 2, 3, 9, 10, and 11 can specifically interact with ASK1 and may have an overlapping function. Since AtTLP5, 7, and 8 cannot interact with any ASK, it is suggested that they may have lost their original function(s) and/or acquired new function(s) during evolution.

並列關鍵字

Arabidopsis ； ASK ； F-box domain ； F-box protein ； Protein-protein ； Interaction ； Ttubby domain ； TULP ； Yeast two-hybrid