Biochemical and physiological characterization of PIN-FORMED auxin efflux carriers

植物激素生长素几乎控制植物生长和发育的方方面面。生长素的许多作用是由于化合物的极性运输造成的。PIN形成的生长素出口蛋白(PIN)家族在这一过程中起着关键作用。长期以来，人们对PIN的生化特性知之甚少。我们已经建立了非洲爪哇卵母细胞的生长素外流检测方法，使我们能够全面地描述这个转运蛋白家族的运输特性。我们能够证明不同的PIN之间生长素运输的动力学是不同的，并且所谓的典型PIN的动力学参数依赖于激酶。我们还能够证明，规范PIN的环域不仅是调节的，而且对传输特性也有贡献。最后，我们可以证明无嗜性Pin2突变体的互补不仅取决于PIN的定位，还取决于它们的运输性质。在目前的方案中，我们希望利用非洲爪哇表达系统来进一步鉴定PIN蛋白，并将我们的努力扩展到所谓的非规范PIN，其中环区显著减少。我们将确定哪些环结构域带来PIN蛋白的独特性质，并确定哪些环结构域是对激酶的差异反应所必需的。为了更详细地了解转运蛋白和激酶之间的相互作用，我们将使用酵母、两种杂交试验和几种不同的生化方法来进一步表征环和激酶之间的相互作用。然后，我们将使用PIN突变表型的互补来验证来自Planta中的生化分析的预测。最后，我们将描述PIN之间的相互作用以及PLANTA中生理上相关的PIN-激酶对。这些研究的结果将大大增加我们对PIN介导的生长素运输发生的作用模式的理解。

The phytohormone auxin controls virtually all aspects of plant growth and development. Many of the effects of auxin are due to the polar transport of the compound. The PIN-FORMED (PIN) family of auxin exporters is crucial in this process. The biochemical properties of PINs were for a long time poorly understood. We have established an auxin efflux assay from Xenopus oocytes that allowed us to comprehensively characterize the transport properties of this transporter family. We were able to show that the kinetics of auxin transport differ between different PINs and that the kinetic parameters of the so-called canonical PINs are dependent on the kinase. We were also able to show that the loop domain of canonical PINs is not merely regulatory, but it contributes to the transport properties. Finally, we could show that complementation of the agravitropic pin2 mutant depends not only on the localization of PINs but also on their transport properties. In the present proposal we wish to take advantage of the Xenopus expression system to further characterize PIN proteins and extend our efforts to the so-called non-canonical PINs in which the loop domain is significantly reduced. We will determine which loop domains bring about unique properties of PIN proteins and identify loop domains which are required for the differential response to kinases. We will further characterize the interaction between loop and kinases using yeast two hybrid assays and several different biochemical approaches with the aim to understand the interplay between transporter and kinase in more detail. We will then verify predictions derived from the biochemical analyses in planta using the complementation of pin mutant phenotypes. Finally, we will characterize the interaction of PINs as well as physiologically relevant PIN-kinase pairs in planta. The results of these studies will substantially increase our understanding of the mode-of-action by which PIN-mediated auxin transport occurs.