硝酸鹽轉運蛋白CHL1與氫離子幫浦AHA2的交互作用

The interaction between nitrate transporter CHL1 and proton pump AHA2

許為綺(Wei-Chi Syu)

指導教授 ： 蔡宜芳

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.00259

摘要

CHL1是一個協同運輸的硝酸鹽轉運蛋白，它透過氫離子驅動力的協助來運輸硝酸鹽。此外，CHL1還是一個雙親和性蛋白，會依據環境中硝酸鹽濃度而改變其對硝酸鹽的親和性。本實驗室先前酵母菌雙雜合篩選中，發現CHL1會與氫離子幫浦AHA2互相結合。已知AHA2是一個能創造氫離子驅動力的自我抑制型幫浦蛋白，需要其它激酶或去磷酸酶對AHA2蛋白質C端R domain進行磷酸化或去磷酸化修飾，才可以調控其氫離子幫浦活性。本研究針對CHL1在運輸硝酸鹽上對氫離子的需求，以及CHL1與AHA2的結合進行分析。爪蟾卵硝酸鹽吸收實驗結果顯示，在植物生理pH值範圍內，CHL1在高親和性模式下，硝酸鹽吸收能力隨pH值下降而上升；在低親和性模式下，吸收能力幾乎不受pH值影響。以酵母菌雙雜合實驗分析CHL1與AHA2的結合，發現不同親和性模式下的CHL1與AHA2的結合強度沒有差異。而CHL1除了能與AHA2結合之外，還可以與同一家族的AHA1、4、11互相結合。分析CHL1與AHA2的不同片段區域之結合強度，發現CHL1與AHA2的R domain結合能力非常微弱，而對AHA2之第七到第十個穿膜蛋白的結合能力最強，但若第七到第十個穿膜蛋白，加上最末端R domain，則結合能力會下降，推測R domain會阻礙CHL1與AHA2的結合。我們亦使用電腦模擬CHL1與AHA2可能的結合方式，並與酵母菌雙雜合試驗結果進行比較，推論出最有可能之結果。此外，也發現能調控CHL1親和性轉換的CIPK23與ANI皆會與AHA2結合，且這樣的結合需要R domain的存在，推測它們能夠調控AHA2的活性。但是，我們以RS-72互補實驗發現，CHL1、CIPK23皆無法獨自活化AHA2的氫離子幫浦活性。我們推測，CHL1可能需要與CIPK23或其他蛋白共同合作，才有辦法對AHA2進行調控。

關鍵字

CHL1 ； AHA2 ； 協同運輸 ； 吸收硝酸鹽 ； 氫離子驅動力

並列摘要

CHL1, using proton gradient as driving force to transport nitrate, is a nitrate co-transporter. Moreover, it is a dual-affinity transporter that can switch nitrate affinity according to the nitrate concentrations in the environment. Previous study in our lab found that CHL1 can interact with AHA2, a H+-ATPase, in a yeast two-hybrid screen. AHA2 generates the proton motive force but is an auto-inhibited proton pump. It needs kinase and phosphatase to modified the C-terminal R domain to regulate the pump activity. In this study, I focus on the requirement of proton motive force on CHL1 nitrate uptake and the interaction between CHL1 and AHA2. In the oocyte nitrate uptake study, we found that within the plant physiological pH range, high-affinity transport activity of CHL1 showed more dramatic difference between low pH and high pH condition; while the low-affinity transport activity of CHL1 show little or not difference between low- and high-pH condition. Yeast two-hybrid study showed that AHA2 can interact both CHL1 T101A and CHL1 T101D suggesting that CHL1 at both high- and low-affinity modes can interact with AHA2. CHL1 can also interact with other AHA family member including AHA1, AHA4 and AHA11. Analyzing the interaction between CHL1 and different truncated forms of AHA2, we found the interaction between CHL1 and AHA2 R domain is very weak, and the AHA2 transmembrane domain 7 to 10 has the strongest interaction with CHL1. But the R domain hampered the interaction between CHL1 and AHA2 as AHA2 transmembrane domain 7 to 10 with R domain interact with CHL1 weaker than the one without R domain. We also use computer to simulate the interaction model between CHL1 and AHA2 to predict the potential model that can accommodate yeast two-hybrid results. We also found that CIPK23 and ANI, known to regulate the CHL1, can interact with AHA2 and their interaction with AHA2 requires the presence of R domain, suggestion that they might regulate the activity of AHA2. But using RS-72 complementation assay, we found that CHL1 and CIPK23 cannot activate the pump activity of AHA2. More study is required to find out if CHL1 need to cooperate with CIPK23 or other proteins to regulate AHA2.

並列關鍵字

CHL1 ； AHA2 ； Cotransport ； Nitrate uptake ； proton motive force

參考文獻

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