Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin
Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin
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DOI：
10.1074/jbc.m110.135657
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Roberts, Daniel M.
中科院分区：
文献类型：
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作者：
Masalkar, Pintu;Wallace, Ian S.;Roberts, Daniel M.
根瘤素26 （nod26）是一种重要的内在蛋白，是大豆根瘤共生体膜（SM）上的主要蛋白质成分。在功能上，nod26为水、渗透物和NH3在SM中形成了一个低能量的运输途径。除了运输功能外，新出现的证据表明，膜上高浓度的主要内在蛋白为各种细胞质蛋白提供了相互作用和对接的靶点。研究表明，nod26的c端结构域肽与大豆根瘤提取物中的一个40 kda蛋白相互作用，质谱鉴定该蛋白为大豆细胞质谷氨酰胺合成酶GS(1) β 1。荧光光谱分析表明，重组大豆GS(1) β 1以1:1的化学计量（K-d = 266 nM）结合nod26 c -末端结构域。GS(1) β 1也与分离的SMs结合，这种结合可以通过与nod26的c端肽预孵育来阻断。利用分裂泛素酵母双杂交系统或双分子荧光互补进行的体内实验表明，大豆根瘤中表达的四种细胞质GS亚型与全长根瘤相互作用26。主要的氨同化酶GS与NH3转运体nod26的保守c端结构域结合，通过将酶定位到共生体膜的胞质侧，促进固定氮的有效同化，并防止潜在的氨毒性。
Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N-2-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH3 across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS(1)beta 1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS(1)beta 1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K-d = 266 nM). GS(1)beta 1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH3, is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.