Multiple Complexes of Nitrogen Assimilatory Enzymes in Spinach Chloroplasts: Possible Mechanisms for the Regulation of Enzyme Function

Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

氮同化是维持植物生长与生产力的核心生物学过程。本研究通过蓝Native电泳（BN-PAGE）结合蛋白质印迹（Western blots）分析证实：菠菜叶绿体中参与氮同化的三种叶绿体酶——谷氨酸合酶（GOGAT）、亚硝酸还原酶（NiR）与谷氨酰胺合成酶（GS），可分别组装为不同的蛋白质复合物。在叶绿体基质组分中，GOGAT与NiR不仅以单体形式存在，还分别形成了分子量730 kDa的新型单一尺寸复合物，以及多种大于120 kDa的复合体型貌。经BN-PAGE分离后，这些复合物在二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（2D SDS-PAGE）中均呈现出与对应单体一致的迁移率。当叶绿体基质溶剂的pH发生变化时，含GOGAT的730 kDa复合物可解离为单体，而NiR的多种复合体型貌则可逆地转化为单体形式。另一方面，抗GS抗体检测到的信号不仅存在于基质中，对应传统的420 kDa十聚体全酶复合物；同时在类囊体中还存在一种新型的560 kDa复合物。在2D SDS-PAGE分析中，560 kDa复合物中的多肽链迁移速率慢于420 kDa复合物的多肽，这提示该复合物可能由不同的GS同工型组装而成，或是同一GS蛋白发生了翻译后修饰。本研究通过Native条件下的凝胶内GS活性检测，以及NiR、GOGAT与其生理电子供体铁氧还蛋白（ferredoxin）的结合能力，评估了这些多复合物的功能。结果表明，这三种氮同化酶的尺寸与定位多样性，可能与其酶功能的生理调控相关，这与近期报道的碳同化酶的调控机制类似。