Tobacco RhoGTPase ACTIVATING PROTEIN1 spatially restricts signaling of RAC/Rop to the apex of pollen tubes
Tobacco RhoGTPase ACTIVATING PROTEIN1 spatially restricts signaling of RAC/Rop to the apex of pollen tubes
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DOI：
10.1105/tpc.106.045336
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发表时间：
2006-11-01
期刊：
影响因子：
11.6
通讯作者：
Kost, Benedikt
中科院分区：
文献类型：
--
作者：
Klahre, Ulrich;Kost, Benedikt
Rho型小GTP酶如RAC 5对烟草花粉管极性的维持具有重要作用。我们先前表明RhoGDI 2是RAC 5定位所必需的。在这里，我们描述了GTdR激活蛋白RhoGAP 1，它控制着RAC 5的活性区域。RhoGAP 1 N-末端和CRIB（用于Cdc 42/Rac相互作用结合）结构域都是将黄色荧光蛋白-RhoGAP 1融合物靶向靠近但不在花粉管顶端的质膜所必需的。我们认为，这种定位限制顶端Rho型GTdR活性向侧翼扩散，这确保了RAC信号在顶端的维持。CRIB结构域不是必需的，但增强了RhoGAP 1对花粉管特异性RAC 5的体外活性。RhoGAP 1的GAP活性降低突变导致类似于过表达RAC 5的花粉管的气球状花粉管。为了确定RhoGAP 1的特异性靶向机制，我们分离了与RhoGAP 1相互作用的14-3-3蛋白。当与RhoGAP 1过表达时，它抵消了RhoGAP 1过表达的生长阻滞作用并减弱了RhoGAP 1膜定位，但单独过表达仅诱导小的结构变化。我们建议，灭活RAC 5的subapically本地化的RhoGAP 1，连同动态重新定位的灭活RAC 5从侧翼到顶端的RhoGDI 2，导致空间限制RAC 5的花粉管顶端，从而维持极性生长。
Regulation by Rho-type small GTPases, such as RAC5, is important for the maintenance of polarity in tobacco (Nicotiana tabacum) pollen tubes. We previously showed that RhoGDI2 is necessary for RAC5 localization. Here, we describe the GTPase activating protein RhoGAP1 that controls the area of RAC5 activity. RhoGAP1 N-terminal and CRIB (for Cdc42/Rac-interactive binding) domains are both necessary for targeting yellow fluorescent protein-RhoGAP1 fusions to the plasma membrane close to, but not in, pollen tube apices. We propose that this localization restricts apical Rho-type GTPase activity from spreading toward the flanks, which ensures the maintenance of RAC signaling at the apex. The CRIB domain is not required but enhances in vitro RhoGAP1 activity toward the pollen tube-specific-RAC5. A mutation reducing GAP activity of RhoGAP1 leads to ballooning pollen tubes resembling those overexpressing RAC5. To ascertain the specific targeting mechanism of RhoGAP1, we isolated a 14-3-3 protein interacting with RhoGAP1. When overexpressed with RhoGAP1, it counteracts the growth-retarding effect of RhoGAP1 overexpression and attenuates RhoGAP1 membrane localization but, overexpressed alone, induces only small architectural changes. We propose that inactivation of RAC5 by the subapically localized RhoGAP1, together with dynamic relocalization of inactivated RAC5 from flanks to tip by RhoGDI2, leads to spatial restriction of RAC5 to pollen tube apices, thereby sustaining polar growth.