Intermolecular and Intramolecular Interactions of the Arabidopsis Plasma Membrane Proton Pump Revealed Using a Mass Spectrometry Cleavable Cross-Linker
Intermolecular and Intramolecular Interactions of the Arabidopsis Plasma Membrane Proton Pump Revealed Using a Mass Spectrometry Cleavable Cross-Linker
复制标题
使用质谱可裂解交联剂揭示拟南芥质子泵的分子间和分子内相互作用
DOI：
10.1021/acs.biochem.0c00268
复制
发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Sussman, Michael R.
中科院分区：
文献类型：
--
作者：
Nguyen, Thao T.;Blackburn, Matthew R.;Sussman, Michael R.
在植物和真菌中，质膜质子泵（H+-ATPase）在质膜上建立电化学梯度，作为离子和营养物质跨细胞膜二次运输的驱动力。这是一种必需的酶，在许多重要的过程中发挥作用，包括气孔运动，细胞伸长和细胞对激素，光和其他环境条件刺激的反应。因此，了解H+-ATP酶的活性是如何调节的，对于了解植物如何适应不同的生长条件非常重要。H+-ATP酶的C-末端调节结构域的自抑制作用已得到充分证实，并被认为是通过与催化结构域的相互作用介导的。在这里，使用赖氨酸反应性质谱裂解交联剂DSSO，我们发现的C-末端结构域的thetiopsis H +-ATP酶2（AHA 2）广泛交联的致动器，核苷酸结合，和磷酸化结构域，这表明C-末端结构域调节催化循环，通过调节这些结构域的相对位置。有趣的是，在预测的质子结合位点（TM 6中的Asp-684）附近发生了几个C末端交联，这表明C末端结构域可能调节质子流出。此外，在SDS-PAGE上解析的单体蛋白中检测到AHA 2的C-末端结构域和其他结构域之间的交联，这表明分子内相互作用也可能参与酶活性的调节。最后，我们观察到14 N-AHA 2（未标记）和15 N-AHA 2（标记）的C-末端结构域与其他结构域之间的混合同位素交联，支持我们的模型，即寡聚H+-ATP酶可能以“头-尾”构型自抑制相邻单体。
In plants and fungi, the plasma membrane proton pump (H+-ATPase) establishes an electrochemical gradient across the plasma membrane, which serves as the driving force for the secondary transport of ions and nutrients across the cell membrane. This is an essential enzyme that functions in many important processes including stomatal movement, cell elongation, and cellular responses to stimuli from hormones, light, and other environmental conditions. Therefore, understanding how the activity of the H+-ATPase is regulated is important to understand how plants adapt to different growth conditions. The autoinhibitory effect of the C-terminal regulatory domain of H+-ATPase is well-established and is thought to be mediated by interactions with the catalytic domains. Here, using the lysine reactive mass spectrometry cleavable cross-linker DSSO, we found that the C-terminal domain of theArabidopsisH+-ATPase 2 (AHA2) cross-linked extensively with the actuator, nucleotide-binding, and phosphorylation domains, suggesting that the C-terminal domain regulates the catalytic cycle by modulating the relative positions of these domains. Interestingly, several C-terminal cross-links occurred near a predicted proton binding site (Asp-684 in TM6), suggesting that the C-terminal domain may regulate proton efflux. Additionally, cross-links between the C-terminal domain and other domains of AHA2 were detected in a monomeric protein resolved on SDS-PAGE, suggesting that intramolecular interactions may also be involved in the regulation of enzyme activity. Finally, we observed mixed-isotope cross-linking between the C-terminal domain and other domains of14N-AHA2 (unlabeled) and15N-AHA2 (labeled), supporting our model that oligomeric H+-ATPase may autoinhibit the neighboring monomer in a “head-to-tail” configuration.