The interaction between nitrate transporter CHL1 and proton pump AHA2
Date Issued
2015
Date
2015
Author(s)
Syu, Wei-Chi
Abstract
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.
Subjects
Cotransport
Nitrate uptake
proton motive force
Type
thesis
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