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標題: 冰花鹽誘導 mcSKD1 蛋白 ATPase 活性及組織表現專一性分析
Analyses of ATPase activity and tissue-specific expression of a salt-induced ice plant protein mcSKD1
作者: 姜智彬
Chiang, Chih-Pin
關鍵字: mcSKD1;冰花;ATPase activity;ice plant
出版社: 生命科學系
mcSKD1為耐鹽植物冰花(Mesembryanthemum crystallinum L.)之鹽誘導基因,本論文主要是探討mcSKD1蛋白在冰花耐鹽機制中所扮演的角色。mcSKD1蛋白胺基酸序列與老鼠 SKD1 (suppressor of K+ transport growth defect) 及酵母菌 VPS4 (vacuolar protein sorting) 蛋白有很高的相似性,三者皆具有一段高度保留的 ATPase (or AAA) domain,屬於 AAA-type (ATPase associated with a variety of cellular activities) ATPase 蛋白家族。由酵母菌互補測試中發現,老鼠 SKD1 與冰花 mcSKD1 蛋白皆具有幫助鉀離子吸收的功能,此外 mcSKD1 蛋白還與鈉離子的區隔有關。而 酵母菌VPS4 蛋白已被證實具有 ATPase 酵素活性,並參與酵母菌液泡蛋白的運輸,在 endosomal transport 系統中扮演重要的角色。為了探討 mcSKD1 蛋白於冰花細胞內的功能與耐鹽機制的關係,本論文利用大腸桿菌大量表現重組 mcSKD1 蛋白,並以親合性管柱純化,分析其 ATPase 酵素活性及蛋白特性,測得酵素活性 Km = 2.73 mM、Kcat = 60.32mol/min/g,經由酵素親合性分析與 nucleotide binding assay,發現在高濃度 ATP 下,ADP 會與 mcSKD1 蛋白緊密結合,導致 ATPase 酵素活性的降低。此外利用 K177A、E231Q、K177A/E231Q 等定點突變探討高度保留性序列 Walker A、B motifs 對 mcSKD1 酵素活性的影響,發現突變株與野生株的相對酵素活性分別為 K177A:41 %、E231Q:14 %、K177A/E231Q:160 %,顯示 Walker A、B motifs 在水解 ATP 的反應中扮演重要的角色。
以專一性抗體偵測 mcSKD1 蛋白於冰花各器官的累積情形,在加鹽及未加鹽處理的葉、根,花與種子中皆可偵測到 mcSKD1 蛋白的累積,顯示 mcSKD1 為持續表現之蛋白。利用免疫螢光法偵測 mcSKD1 蛋白的組織表現專一性,發現 mcSKD1 蛋白在根的皮層及韌皮部、葉的腎型細胞、花粉囊與發育中的種皮內累積,推測與冰花生殖細胞生長發育時的營養吸收及鹽逆境下鈉離子的區隔相關。此外由蔗糖梯度離心與免疫金標定法發現,mcSKD1 蛋白分佈於密度很輕的microsome fractions,且聚集形成small vesicles,顯示 mcSKD1 可能與 VPS4 蛋白具有類似的功能,參與蛋白經由內膜系統運輸之過程(endomembrane protein trafficking)。根據以上結果推測,mcSKD1 蛋白在鹽逆境下,利用其 ATPase 酵素活性,提高液泡蛋白運輸之效率,在腎型細胞中參與鈉離子的區隔,協助根部鉀離子的吸收,以調節滲透潛勢,降低鹽逆境所造成的傷害。

A salt-induced gene mcSKD1 was identified from halophyte Mesembryanthemum crystallinum L. (ice plant). The main focus of this thesis is to examine the roles of mcSKD1 protein participate in the salt-tolerant mechanism of ice plant. The amino acid sequence of mcSKD1 was highly homologous to mouse SKD1 (suppressor of K+ transport growth defect) and yeast VPS4 (vacuolar protein sorting). All of them have a highly conserved ATPase (or AAA) domain, belong to AAA-type (ATPase associated with a variety of cellular activities) ATPase protein family. Yeast complementation assay showed mcSKD1 and mouse SKD1 were involved in K+ uptake and mcSKD1 was responsible for the compartmentation of Na+. VPS4 was confirmed with vacuolar protein sorting of endosomal transport system through the ATPase activity. To examine the cellular function of mcSKD1 and roles in salt-tolerant mechanism in ice plant, recombinant mcSKD1 was over-expressed in E. coli and purified by affinity column to analyze ATPase activity and enzymatic properties of protein. The enzyme kinetics showed mcSKD1 has ability to hydrolyze ATP with a Km of 2.73 mM and a Kcat of 60.32mol/min/g. Use the affinity- and nucleotide-binding assay, we found the ATPase activity was greatly reduced by ADP tightly bound with mcSKD1 in high ATP concentrations. The structure of ATPase domain was further examined by point mutation of highly conserved Walker A and B motifs. Three mutants were constructed, namely K177A, E231Q and K177/E231Q. The wild-type and mutants' relative ATPase activity were K177A: 41%, E231Q: 14 % and K177A/E231Q: 160 %. The results indicated that the Walker A and B motifs were important to ATP hydrolysis ability of mcSKD1.
Accumulation of mcSKD1 at different organs was detected by anti-mcSKD1 antibody. We found mcSKD1 accumulated in leaves, roots, flowers and seeds of unstressed and stressed plants. The results indicated mcSKD1 was constitutively expressed protein. The immune-fluorescence was used to detect the tissue-specific expression of mcSKD1. The mcSKD1 was specifically accumulated in cortex and phloem of root, bladder cells of leaves, pollen sac and developing seed coat. The results suggested mcSKD1 plays a role in nutrient uptake of developing reproductive cells and responsible for the compartmentation of excess Na+ under salinity stress. Furthermore, the accumulation of mcSKD1 was found to localize at low-density small vesicles by gradient ultra-centrifugation and immuno-gold labeling. The results indicated mcSKD1 had similar function with VPS4, which was involved in cellular protein sorting. In conclusion, the ATPase activity of mcSKD1 drives the energy obtained by ATP hydrolysis and uses in compartmentation of excess Na+ in bladder cells and K+ uptake in roots, and as the result, the ion homeostasis and osmotic potential was maintained.
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