以酵母菌雙雜合系統篩選阿拉伯芥中與 AtMAPRs 有交互作用之蛋白質

Abstract

我們在阿拉伯芥中發現四個與豬的 MAPR (membrane associated progesterone receptor conponent 1) 有同源性的蛋白質,並命名為 AtMAPR2、AtMAPR3、 AtMAPR4 與 AtMAPR5。雖然對此四個蛋白質的功能缺乏全面性的了解,但我們 推測 AtMAPRs 參與調控環境中訊息的接收,例如植物本身分泌的荷爾蒙、光照或 逆境。本實驗室先前的酵母菌雙雜合實驗結果顯示,AtMAPRs 可能與聚泛素 (polyubiquitin)、MYB3 轉錄因子、GTP 結合蛋白質 (AtTOC33) 以及丙胺酸-乙醛酸 轉胺? (alanine-glyoxylate aminotransferase,AGT1)。然而,AtMAPR3、AtMAPR4 與 AtMAPR5 所含有的推測性穿越膜區塊 (putative transmembrane domain) 可能影響 酵母菌雙雜合的結果。因此,我們使用除去一到四十個胺基酸的 AtMAPR5 當成釣 餌,透過酵母菌雙雜合系統來篩選主要以阿拉伯芥花製成的 cDNA 庫 (CD4-30)。 在二十六個分別可能與 AtMAPR5 有交互作用的蛋白質中,推測 C3HC4-type RING 蛋白質 (At3g58030) 與一種 FKBP-type peptidyl-prolyl cis-trans isomerase (AtFKBP16-2; At4g39710) 可能分別直接與 AtMAPR5 在阿拉伯芥中一同發揮其效 能。根據這兩個可能與 AtMAPR5 產生交互作用的蛋白質,我們提出兩套模型來解 釋 AtMAPRs 可能的作用機制:(一) C3HC4-type RING 蛋白質可能具有 E3 ligase的 活性並且藉者調控 AtMAPRs 的降解而達到傳遞訊息的功能。(二) AtMAPRs 可能 於光合作用 (photosynthesis) 中與 AtFKBP16-2 一同扮演電子傳遞者的角色。許多工 作仍須繼續進行來證明此二假設的真實性。簡言之,酵母菌雙雜合系統的結果提供 許多資訊讓我們對 AtMAPRs 的功能做進一步的推測。

Four AtMAPRs (AtMAPR2, AtMAPR3, AtMPAR4, and AtMAPR5) sharing 30- 40% similarity with porcine MAPR (membrane associated progesterone receptor component 1) have been identified in Arabidopsis. The function of the four AtMAPRs is not clear, but we speculated that they could participate in regulating environmental stimuli such as plant hormones, light, or stresses. Previous yeast two-hybrid assay indicated that AtMAPRs might interact with polyubiquitin, MYB3 transcription factor, AtTOC33 (GTP-binding protein), and AGT1 (alanine-glyoxylate aminotransferase). However, the presence of putative transmembrane domain of AtMAPR3, AtMAPR4, and AtMAPR5 might interfere with yeast two-hybrid results. In this work, we used truncated AtMAPR5, lacking the 1-40 amino acid residues, as bait to screen flower two-hybrid cDNA library, CD4-30. Among the 26 different interacting proteins obtained from the screening, a C3HC4-type RING protein (At3g58030) and a FKBP-type peptidyl-prolyl cis-trans isomerase, AtFKBP16-2 (At4g39710) was perhaps to function together with AtMAPR5 in Arabidopsis. We proposed two models based on these two interacting proteins that: (1) C3HC4-type RING protein might function as E3 ligase and promote the degradation of AtMAPRs. (2) AtMAPRs might be involved in electron transferring in photosynthesis with AtFKBP16-2. Further work has to be done to testify these two assumptions. Briefly, these yeast two-hybrid results provided hints to the function of AtMAPRs.