Clathrin adaptor EPSIN1 ( EPS1 ) modulates plasma membrane abundance of PLEIOTROPIC DRUG RESISTANCE PDR9 for effective hormone homeostasis

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The cellular machinery that directs the indole-3-butyric acid (IBA) effluxer PLEIOTROPIC DRUG RESISTANCE 9 (PDR9) to the outer plasma membrane (PM) domain of the root epidermis remains poorly understood. This study combines biochemical fractionation, live-cell imaging, and physiological assays to identify the trans-Golgi Network-localized clathrin adaptor EPSIN1 (EPS1) as a novel modulator of PDR9 abundance at the root PM for IBA-dependent physiological processes. These findings expand EPS1's roles from plant immunity to hormone homeostasis, thereby advancing the limited understanding of biological functions for plant EPSINs. Despite its key role at the root–soil interface, the cellular machinery that directs plasma membrane (PM) proteins, such as the indole-3-butyric acid (IBA) effluxer PLEIOTROPIC DRUG RESISTANCE 9 (PDR9), to the outer root epidermis remains poorly understood. This study identified the clathrin adaptor EPSIN1 (EPS1), localized to the trans-Golgi Network/early endosome (TGN/EE), as a novel modulator of PDR9 PM abundance and IBA-dependent physiological processes, thereby expanding the limited understanding of biological roles of plant EPSIN family members. In plants, the protein composition of the PM plays a critical role in mediating interactions between the cell and its environment, enabling cells to mount appropriate responses to biotic and abiotic stresses and processes related to growth and development. To establish and maintain the correct spatiotemporal abundance of proteins in the PM, plant cells utilize clathrin-coated vesicles (CCVs) that form at the TGN/EE or the PM to traffic proteins to or from the PM, respectively (Ekanayake et al., 2019; Dahhan & Bednarek, 2022; Johnson, 2024). Clathrin cannot directly bind to lipids or cargo proteins. Instead, CCV-mediated trafficking relies on specific clathrin adaptor and accessory proteins to coordinate CCV formation, release, and uncoating at the donor membrane for subsequent fusion with the correct target membrane. Despite advances in identifying CCV components and their roles in clathrin-mediated trafficking, the cargo proteins linked to specific plant CCV components and their roles in downstream physiological responses remain largely unknown. EPSINs (EPSs) are monomeric clathrin adaptors that utilize their Epsin N-terminal Homology (ENTH) domain to bind to specific lipids as well as peptide motifs in their unstructured C-terminus to link clathrin core and adaptor proteins to the donor membrane site for CCV formation. Compared with animal and yeast EPSINs, relatively little is known about plant EPSINs and their cellular roles in physiological responses. The model plant Arabidopsis thaliana (Arabidopsis) encodes seven ENTH-domain proteins (Zouhar & Sauer, 2014; Collins et al., 2020; Feng et al., 2022), including EPS1 localized at the TGN/EE, where it colocalizes with clathrin and the multimeric adapter protein AP-1 (Song et al., 2006; Heinze et al., 2020). Notably, EPS1 but not MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) – the other known TGN/EE-localized Arabidopsis Epsin (Heinze et al., 2020) – contributes to effective resistance to the leaf pathogenic bacteria Pseudomonas syringae pv tomato (Pto) DC3000 (Collins et al., 2020; Mason et al., 2023). Specifically, EPS1 positively modulates plant immune signaling and pattern-triggered immunity because in eps1 mutants, impaired immune responses correlate with reduced accumulation of the immune receptor FLAGELLIN SENSING2 (FLS2) and the convergent coreceptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) in the PM (Collins et al., 2020). However, EPS1 does not contribute to ligand-induced endocytosis of FLS2 (Collins et al., 2020). To identify potential PM cargo proteins trafficked by EPS1, we previously used biochemical fractionation and quantitative enriched PM proteomics, revealing that EPS1 regulates PM accumulation of a distinct set of structurally diverse proteins with varied cellular functions (Collins et al., 2020). Compared with wild-type (WT), the most significantly decreased protein in the enriched PM proteome of eps1-2 was PDR9, also called ATP-BINDING CASSETTE G37 (ABCG37)/POLAR AUXIN TRANSPORT INSENSITIVE 1 (PIS1) (Ito & Gray, 2006; Ruzicka et al., 2010; Frick & Strader, 2018). PDR9 effluxes IBA, the storage form of the natural plant hormone auxin indole-3-acetic acid (IAA), to promote growth and morphogenesis (Strader et al., 2008; Sauer et al., 2013; Frick & Strader, 2018; Damodaran & Strader, 2019; Cohen & Strader, 2024). Alterations in IBA conversion to IAA result in multiple developmental defects in plants, highlighting the critical role of IBA in maintaining auxin homeostasis (Damodaran & Strader, 2019). In addition to IBA, PDR9 contributes to root exudation of coumarins into the rhizosphere to promote iron (Fe)-mobilization under Fe-deficiency (Fourcroy et al., 2014; Ziegler et al., 2017; Robe et al., 2021). Consistent with these functions, PDR9 is a root-specific transporter localized in the outer lateral domain of epidermal root cells (Langowski et al., 2010; Ruzicka et al., 2010). Requirements for PM localization of PDR9 differs from that of the well-characterized IAA efflux carriers PINFORMED1 (PIN1) and PIN2, as it does not rely on vesicle trafficking components involved in the basal and apical targeting of PIN proteins (Langowski et al., 2010). Moreover, unlike PINs, newly synthesized PDR9 is directly secreted to its functional site at the PM (Langowski et al., 2010; Ruzicka et al., 2010). However, the molecular machinery regulating the PM abundance of PDR9 remains largely unknown. In this study, we use a combination of biochemical fractionation, live-cell imaging, and physiological assays to provide evidence that (a) proper PM abundance of PDR9 requires EPS1 and (b) reduced PM levels of PDR9 in the eps1 mutant are associated with root defects linked to impaired IBA homeostasis. Thus, our findings expand EPS1's roles in physiological processes from plant immunity in leaves to hormone homeostasis in roots. Quantitative proteomic comparisons of enriched PM from whole seedlings identified the IBA efflux carrier PDR9 as the most significantly decreased protein in eps1-2 null mutant compared to WT Col-0 (Collins et al., 2020), implicating the clathrin adaptor EPS1 as a modulator of PDR9 abundance in the PM. Using quantitative real-time polymerase chain reaction (qRT-PCR), we showed that the reduced PM accumulation of PDR9 protein in eps1-2 was not due to changes in transcript levels, as PDR9 mRNA was not significantly different between eps1-2 and Col-0 (Fig. 1a). The pdr9-2 null mutant (Ito & Gray, 2006) served as a negative control, exhibiting minimal PDR9 transcript (Fig. 1a) and no significant detectable PDR9 protein in root microsomal fractions (Fig. 1b, Supporting Information Fig. S1A,B), confirming the specificity of the αPDR9 antibody in immunoblots. In plants, microsomes consist of the PM as well as membranes derived from the ER, Golgi apparatus, TGN/EE, tonoplast and other endosomal compartments. To validate the proteomics results, we adapted the biochemical method using differential centrifugation to obtain microsomes that were treated with the detergent Brij58 (Zhang & Peck, 2011; Collins et al., 2017, 2020; Ekanayake et al., 2021) to isolate enriched PM from Arabidopsis roots (Figs 1c, S1C,D). Fractionation efficacy was verified by immunoblotting using compartment-specific antibodies: the PM fractions for eps1-2 and Col-0 were enriched for the PM marker protein AHA H+-ATPases and PM INTRINSIC PROTEIN (PIP) and depleted for the ER membrane marker CALNEXIN (CNX) (Figs 1c, S1C). While PDR9 accumulation was apparently similar in microsomal fractions (Figs 1b,c, S1C,D), PDR9 levels were lower in PM fractions of eps1-2 than in WT (Figs 1c, S1C), confirming the previous quantitative PM proteomics data from whole seedlings (Collins et al., 2020). These conclusions were supported by quantifying

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