NLS-Motif of EIN2 key regulator: Functional and mechanistic relevance for the ethylene signaling pathway

乙烯信号通路调节植物细胞的重要生长发育过程。内质网膜整合蛋白EIN2是这一通路的中心元件，它将信号从内质网连接到细胞核。虽然普遍接受的EIN2‘裂解和穿梭’模型似乎解释了乙烯信号如何从内质网传递到细胞核，但与ER锚定受体的复合体形成、核质运输和EIN2的核输入有待进一步分析，以全面揭示EIN2作用的分子机制。该项目旨在阐明参与这些过程的途径和结构。我们实验室以前的研究表明，EIN2和EIN2-NLS多肽与乙烯受体家族具有高亲和力结合。现在我们的目标是解决NLS基序在受体上的准确结合位置。有了这些信息，我们将设计功能丧失的突变体，用于植物研究，使我们能够评估EIN2NLS结合在内质网定位的乙烯信号受体上的机制、相关性和意义。根据EIN2中规范的NLS序列和本实验室的初步工作证明EIN2-NLS肽与核转运蛋白受体IMPa7结合，我们认为EIN2的核质转运和核输入是由IMPA-IMPB系统介导的。在我们的项目中，我们将专注于通过各种植物IMPA亚型识别EIN2货物。在此，我们将确定拟南芥IMPA亚型(IMPa1-IMPa9)识别和结合EIN2所需的分子决定因素。为此，我们将对各种IMPA-EIN2络合物进行定量结合研究和结构研究。随后的植物荧光成像研究将回答哪些EIN2结合的IMPA亚型将EIN2货物输入到细胞核，或者哪些亚型可能由于细胞质滞留而抑制核输入，从而切断乙烯信号从内质网到细胞核的连接。这将揭示IMPA家族对乙烯信号可能的重要调节功能。对IMPA和EIN2NLS突变体的研究将证实这一分析。在成功地将EIN2转移到细胞核之后，一个仍然悬而未决的问题是，EIN2通过什么分子基序和机制稳定了细胞核中的EIN3主转录调节因子。我们将解决这个问题，并使用这两种蛋白质的野生型和突变亚域来研究EIN2-EIN3的相互作用。通过这种方法，我们将定义EIN2-EIN3相互作用的核心复合体。基于这些知识，我们的目标是解决这种络合物的结构，以便对控制这种相互作用的分子结构有详细的了解。我们项目的结果有望弥合乙烯信号转导方面的长期知识差距，并阐明信号级联的核质步骤，该步骤将内质网膜上的初始步骤与细胞核中的下游信号事件连接起来。

The ethylene signaling pathway regulates important growth and developmental processes in plant cells. The ER membrane-integral protein EIN2 is a central element in this pathway bridging signaling from the ER membrane to the nucleus. While the general accepted EIN2 ‘cleave and shuttle’ model plausibly explains how the ethylene signal is transmitted from the ER to the nucleus, complex formation with ER-anchored receptors, nucleocytoplasmic transport and nuclear import of EIN2 await further analysis to fully uncover the molecular mechanisms of EIN2 action. The project aims at elucidating pathways and structures involved in these processes. Previous studies in our lab revealed high affinity binding of EIN2 and EIN2-NLS peptides to the ethylene receptor family. Now we aim to resolve the precise binding site of the NLS-motif at the receptor. With this information we will design loss-of-function mutants that will be used in plant studies allowing us to assess the mechanistic relevance and significance of EIN2 NLS-binding at the ER-localized receptors for ethylene signaling. Based on the canonical NLS-sequence in EIN2 and preliminary work of our lab demonstrating binding of an EIN2-NLS peptide at nuclear transport receptor IMPa7, we propose that nucleocytoplasmic transport and nuclear import of EIN2 is mediated by the IMPa-IMPb system. In our project we will focus on EIN2 cargo recognition by the various plant IMPa isoforms. Thereto, we will determine the molecular determinants in Arabidopsis IMPa isoforms (IMPa1-IMPa9) required to recognize and bind EIN2. Thereto, we will apply quantitative binding studies and structural studies of various IMPa-EIN2 complexes. Subsequent in planta fluorescence imaging studies will answer which of the EIN2-binding IMPa isoforms import the EIN2 cargo to the nucleus or which may inhibit nuclear import due to cytoplasmic retention and thereby disconnect ethylene signaling from the ER to the nucleus. This will uncover possible important regulatory functions of the IMPa family for ethylene signaling. Studies on IMPa and EIN2 NLS-mutants will substantiate this analysis. Following the successful transfer of EIN2 to the nucleus a remaining open question still is by which molecular motifs and mechanisms EIN2 stabilizes the EIN3 master transcriptional regulator in the nucleus. We will address this question and study the EIN2–EIN3 interaction using well-defined wild type and mutant subdomains of both proteins. By this approach, we will define the EIN2–EIN3 interacting core complex. Based on this knowledge we aim to solve the structure of this complex in order to get detailed insight into the molecular structures controlling this interaction. The results from our project are expected to close a longstanding knowledge gap in ethylene signal transduction – and shed light on the nucleocytoplasmic step of the signaling cascade that bridges the initial steps at the ER membrane with the downstream signaling events in the nucleus.