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

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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.