In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.
In vivo cross-linking combined with mass spectrometry analysis reveals receptor-like kinases and Ca(2+) signalling proteins as putative interaction partners of pollen plasma membrane H(+) ATPases.
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体内交联与质谱分析相结合揭示了受体样激酶和 Ca(2 ) 信号蛋白作为花粉质膜 H( ) ATP 酶的假定相互作用伙伴
DOI：
10.1016/j.jprot.2014.05.001
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发表时间：
2014
影响因子：
3.3
通讯作者：
Obermeyer
中科院分区：
文献类型：
--
作者：
Pertl-Obermeyer;Schulze;Obermeyer
在植物受精过程中，花粉粒萌发并产生花粉管，花粉管通过花柱组织生长到卵细胞，将两个精子细胞送去受精。在这个过程中，对特定环境条件的适应和男性和女性器官之间的交流是必不可少的，需要感知内部和外部信号，这些信号被转化为试管生长。质膜H+ atp酶为花粉质膜提供养分、离子和水分吸收的能量，其活性直接影响萌发频率和驱动花粉管伸长。通过与多聚甲醛的内交联、交联PM H+ atp酶复合物的免疫亲和纯化以及随后的质谱分析，揭示了百合花粉中PM H+ atp酶的推定相互作用伙伴，它们可能参与细胞内和细胞外信号的感知和转导。主要的相互作用伙伴包括(i)膜定位的受体样激酶（RLKs）与富含亮氨酸的重复RLKs （LRR-RLKs）形成最大的群体，（ii）相互作用的蛋白激酶、磷酸酶、WD-40结构域蛋白和14-3-3蛋白，它们可以转导细胞内磷酸化依赖的信号，（iii）特定的细胞质Ca2 +信号可以通过相互作用的Ca2 +传感器蛋白、钙调蛋白和钙调蛋白样蛋白以及Ca2 +依赖的蛋白激酶来解码。它们都被鉴定为百合花粉中PM H+ atp酶的互作伙伴。这些确定的相互作用伙伴提供了PM H+ atp酶一般的新假设调节机制，特别是调控花粉管生长速度的新见解。此外，优化的实验策略可以应用于其他非模式生物来识别膜蛋白相互作用。由于膜蛋白丰度低、溶解度差，膜蛋白质组学研究仍然具有挑战性。此外，膜蛋白相互作用在非模式生物如长花百合的研究需要一个公正的制备和检测方法。提出的策略是通过使用不同生化技术的组合来确定PM H+ atp酶的推定相互作用伙伴，即。在体内交联中，免疫亲和纯化和质谱法无需基因工程、转化或其他分子生物学技术，可以很容易地转移到其他蛋白质相互作用的研究中。PM H+ atp酶与14-3-3蛋白的相互作用是一种内在控制，证明了所提出策略的适用性和可靠性，而新发现的相互作用伙伴可能表明PM H+ atp酶的新调节机制。
During fertilisation in plants, pollen grains germinate and generate a pollen tube which grows through the style tissue to the egg apparatus delivering the two sperm cells for fertilisation. For this process, adaption to specific environmental conditions and communication between male and female organs are essential, requiring the sensing of internal and external signals which are translated into tube growth. The plasma membrane (PM) H+ATPase energises the pollen plasma membrane for nutrient, ion and water uptake, but additionally, its activity directly affects the germination frequency and drives the elongation of pollen tubes. A combination ofin vivocross-linking withpara-formaldehyde, immunoaffinity purification of cross-linked PM H+ATPase complexes and subsequent mass spectrometry analysis revealed putative interaction partners of the PM H+ATPase of lily pollen, which are possibly involved in the perception and transduction of intra- and extracellular signals. Major interactions partners included (i) membrane-localised receptor-like kinases (RLKs) with the leucine-rich repeat RLKs (LRR-RLKs) forming the largest group, (ii) interacting protein kinases, phosphatases, WD-40 domain proteins and 14-3-3 proteins that may transduce intracellular, phosphorylation-dependent signals and (iii) specific cytosolic Ca2 +signatures may be decoded by interacting Ca2 +sensor proteins, calmodulin and calmodulin-like proteins, and Ca2 +-dependent protein kinases, which were all identified as interaction partners of the PM H+ATPase in lily pollen. These identified interaction partners suggest new putative regulation mechanisms of the PM H+ATPase in general and new insights in regulating pollen tube growth rates in particular. Furthermore, the optimised experimental strategy can be applied to other non-model organisms to identify membrane protein interactions.Biological significanceMembrane proteomics is still very challenging due to the low abundance and poor solubility of membrane proteins. Furthermore, membrane protein interaction studies in a non-model organism likeLilium longiflorumrequire an unbiased preparation and detection approach. The presented strategy to identify putative interaction partners of the PM H+ATPase by using a combination of different biochemical techniques,i.e. in vivocrosslinking, immunoaffinity purification and mass spectrometry without the need of genetic engineering, transformation or other molecular biology techniques can be easily transferred to other protein interaction studies. The well characterised interaction of the PM H+ATPase with regulating 14-3-3 proteins served as an intrinsic control to proof the suitability and reliability of the presented strategy, whilst newly identified interaction partners may indicate novel regulation mechanisms of the PM H+ATPase.
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影响因子：
4.8
作者：
Maudoux, O;Batoko, H;Morsomme, P
通讯作者：
Morsomme, P
百合花粉粒 14-3-3 蛋白在花粉粒萌发和管生长过程中调节质膜 H ATP 酶活性的分子和生理学特征
影响因子：
4.3
作者：
H. Pertl;M. Himly;R. Gehwolf;Rosa Kriechbaumer;D. Strasser;W. Michalke;K. Richter;F. Ferreira;G. Obermeyer
通讯作者：
G. Obermeyer
包含花粉特异性受体样激酶的信号网络中的相互作用
影响因子：
6.6
作者：
Loecke;Fricke;Humpert;Berken
通讯作者：
Berken
影响因子：
7.2
作者：
Rohila, JS;Chen, M;Fromm, ME
通讯作者：
Fromm, ME
影响因子：
4.8
作者：
Justesen, Bo Hojen;Hansen, Randi Westh;Fuglsang, Anja Thoe
通讯作者：
Fuglsang, Anja Thoe