阿拉伯芥 AtMAPRs 之分子特性研究

Molecular Characterization of AtMAPRs in Arabidopsis

高艾玲(Ai-Ling Kao)

指導教授 ： 楊建志

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.10189

摘要

阿拉伯芥中發現四個與豬的MAPR (membrane associated progesterone receptor conponent 1) 有30~40% 相似度的同源性蛋白質，AtMAPR2、AtMAPR3、AtMAPR4與AtMAPR5。研究指出AtMAPR5 (putative membrane steroid binding protein, MSBP1) 位在細胞膜，藉由促進brassinosteroid (BR) 受體走向內噬作用途徑，負向調控其訊息傳導 (Song et al. 2009)。但對於其他AtMAPRs蛋白質的功能仍然缺乏了解。於大腸桿菌中表現AtMAPRs，並分析其蛋白質特性。AtMAPRs之UV-visible光譜顯示AtMAPRs具有血基質蛋白的特性，綜合過去研究，血基質結合能力似乎是MAPRs蛋白質家族的特徵之一。進一步利用EPR光譜研究AtMAPR2與血基質結合模式，結果顯示AtMAPR2與血基質為高自旋度五配位共價結合形式，並利用定位點突變方式找到兩個影響AtMAPR血基質親和力的重要殘基Tyr44與Tyr92。
之前研究發現AtMAPR3與AtMAPR5在胞外具有E3-independent自身泛素化修飾能力，在本論文中，證實AtMAPR2及AtMAPR4在E3不存在的情況下，也可以進行自身泛素化修飾，且其修飾方式為接上一個泛素。序列比對結果顯示AtMAPRs上具有類似UIM (ubiquitini-interacting motif，為泛素結合區塊UBD的一種) 的序列，其位於血基質結合區塊上。血基質的添加會降低AtMAPR2的自身泛素化修飾反應，血基質結合與泛素化修飾作用有競爭的現象，可能由於這兩種修飾作用都發生在AtMAPR2疏水性口袋結構上。即時定量聚合酶鏈鎖反應顯示AtMAPR2的基因表現會受brassinosteroid的刺激而下降，且AtMAPR2基因表現下降的RNAi植株對brassinosteroid的刺激較不敏感，以上結果顯示AtMAPR2具有血基質結合能力且可能參與brassinosteroid訊息傳導或生合成途徑。
以酵母菌雙雜合法找到AtMAPR3的交互作用蛋白AGT1 (alanine glyoxylate transaminase)，AGT1為參與光呼吸作用的重要酵素，本研究藉由GST pull-down與BiFC (bimolecular fluorescence complementation) 分析驗證兩者交互作用的真實性及專一性，且發現血基質結合區塊對其交互作用的發生扮演重要的角色。在AtMAPR3::GUS突變株GUS活性分析中顯示，AtMAPR3的基因表現會受到光照的誘導。不論在洋蔥表皮或阿拉伯芥原生質體的細胞內定位實驗指出，AtMAPR3會位在細胞膜上與囊狀構造 (vesicles) 中。AtMAPR3基因靜默突變株對過氧化氫的處理較為敏感，且在鹽逆境下種子發芽率較野生型高，AtMAPR3基因表現會受到與植物防禦機制相關的植物荷爾蒙 (茉莉酸與水楊酸) 及鹽逆境刺激而增加。綜合以上結果，AtMAPR3可能藉由與AGT1的交互作用而參與光呼吸作用的調控機制。
AtMAPR4基因靜默突變株與基因表現下降之突變株，在強光培養下呈現淡綠色葉子與開花時間較晚之性狀改變，以酵母菌雙雜合法篩選到AtMAPR4的交互作用蛋白質，為屬於位在ER膜上的轉錄因子NTL11與細胞核孔蛋白WIP1，並進一步以BiFC驗證其交互作用之專一性。NTL11基因表現會受到高溫強光而誘導，調控flavonoids生合成基因的表現，促進花青素合成因應強光傷害。WIP1可調控RanGAP1結合上細胞核膜的過程，調控物質進出細胞核，再者AtMAPR4細胞內定位位在細胞核膜與細胞膜等膜系構造上。推測AtMAPR4在細胞內可能藉由泛素化修飾作用，在強光或高溫的情況下，參與NTL11由ER釋放至細胞核的過程，進一步調控flavinoids生合成基因表現。

關鍵字

血基質結合蛋白 ； 自身泛素化修飾 ； 光呼吸 ； 黃酮類

並列摘要

Four AtMAPRs (AtMAPR2, AtMAPR3, AtMPAR4, and AtMAPR5) in Arabidopsis sharing 30-40% similarity with porcine MAPR (membrane associated progesterone receptor component 1) have been identified and investigated. AtMAPR5 (putative membrane steroid binding protein, MSBP1) is a membrane protein and proposed to negatively regulate brassinosteroid signaling by enhancing the endocytosis (Song et al. 2009). However, the functions of other AtMAPRs are still unclear. In this study, the recombinant AtMAPRs were purified from E.coli and their molecular properties were investigated. The UV-visible absorption spectra showed that AtMAPRs were hemoproteins. The heme binding affinity seems to be a common feature of MAPR family proteins in vitro. The EPR spectra of the purified proteins showed that AtMAPR2 bound a heme chromophore in high-spin, five-coordinated type. Site-directed mutagenesis indicated that two Tyr residues, Y44 and Y92, were important for anchoring heme into a hydrophobic pocket.
Previous study on AtMAPR3 and AtMAPR5 showed they had E3-independent autoubiquitination ability in vitro. In this study, AtMAPR2 and truncated AtMAPR4 also possessed monoubiquitination ability without E3 ligase in vitro. The putative UIM (ubiquitin-interacting motif, a member of UBDs) was located in conserved heme/steroid binding domain of AtMAPRs. The autoubiquitination ability of AtMAPR2 seems to be decreased by adding heme in vitro. Heme binding might compete with the ubiquitin modification owing to their binding to the same hydrophobic pocket in AtMAPRs.
The expression of AtMAPR2 was down-regulated by brassinosteroid as revealed by the real-time PCR analysis and the knockdown mutant of AtMAPR2 showed less sensitivity to brassinosteroid. These results indicated that AtMAPR2 had heme binding affinity in vitro and might be involved in brassinosteroid signaling or synthesis pathway.
By GST pull-down and bimolecular fluorescence complementation (BiFC) assay, AtMAPR3 showed protein-protein interaction with alanine glyoxylate transaminase (AGT1) which participated in the plant photorespiration. The heme/steroid binding domain of AtMAPR3 played an important role in the interaction between AtMAPR3 and AGT1. In AtMAPR3::GUS plants, the GUS activity was up-regulated by light. AtMAPR3-GFP was located in plasma membrane and vesicles in onion epidermal cells or Arabidopsis protoplasts. The AtMAPR3 knockout mutant was more sensitive to H2O2 in 16-day-old seedlings and more resistant to salt stress by seed germination bioassay. The gene expression of AtMAPR3 was increased by treatments with defense-related hormones, jasmonate and salicylic acid, and NaCl. These results revealed that AtMAPR3 might be involved in the regulation on photorespiration by interacting with AGT1.
The knockout and knockdown mutants of AtMAPR4 showed pale green leaves under strong light intensity and longer flowering time. Two interacting protein candidates of AtMAPR4, NTL11 (NTM1-like protein) and WIP1 (WPP-domain interacting protein), were found using yeast two hybrid experiments. The specific interaction between AtMAPR4 and NTL11 was confirmed by BiFC assay. NTL11 is involved in flavonoids biosynthesis. These results showed that AtMAPR4 might regulate flavonoids synthesis by interacting with NTL11.

並列關鍵字

hemoprotein ； autoubiquitination ； photorespiration ； flavonoids

參考文獻

Anzi, C., Pelucchi, P., Vazzola, V., Murgia, I., Gomarasca, S., Piccoli, M. B. &Morandini, P. (2008). The proton pump interactor (Ppi) gene family of Arabidopsis thaliana: expression pattern of Ppi1 and characterisation of knockout mutants for Ppi1 and 2. Plant Biol (Stuttg) 10(2): 237-249.

Baba, K., Nakano, T., Yamagishi, K. &Yoshida, S. (2001). Involvement of a nuclear-encoded basic helix-loop-helix protein in transcription of the light-responsive promoter of psbD. Plant Physiol 125(2): 595-603.

Bartalesi, I., Bertini, I., Ghosh, K., Rosato, A. &Turano, P. (2002). The unfolding of oxidized c-type cytochromes: the instructive case of Bacillus pasteurii. J Mol Biol 321(4): 693-701.

Berry, E. A. &Trumpower, B. L. (1987). Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal Biochem 161(1): 1-15.

Bonza, M. C., Fusca, T., Homann, U., Thiel, G. &De Michelis, M. I. (2009). Intracellular localisation of PPI1 (proton pump interactor, isoform 1), a regulatory protein of the plasma membrane H(+)-ATPase of Arabidopsis thaliana. Plant Biol (Stuttg) 11(6): 869-877.