Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity

Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein complexes and the scaffolding molecule clathrin. Two adaptor protein complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric Adaptor Protein 2 (AP-2) complex and the octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promotor, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth and gravitropic response, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants

Nuevos blancos en la respuesta vegetal a la deficiencia de boro: N-glicosilación de proteínas y la regulación del desarrollo de la raíz)

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 22-01-2016Esta tesis tiene embargado el acceso al texto completo hasta el 22-07-201

Comparative analysis of auxinic compounds transporter ABCG37 and various polarly localized proteins in respect of secretion, trafficking and plasma membrane dynamics

Plants as sessile organisms evolved a specific body structure and at the cellular level mechanisms that allow to survive under extreme environmental conditions. The body shape and subcellular processes are largely dependent on coordinated activity of a small molecule indole-3-acetic acid (IAA), auxin. Local gradients of IAA correlate spatiotemporally with such developmental events like embryogenesis, phyllotaxis, organ initiation or tropisms. Auxin maxima and minima are mostly mediated by auxin efflux carriers PIN's. Asymmetric distribution of these proteins determines the directional flow and facilitates the auxin gradient formation. Aberrations in apical or basal auxin-carriers localisation leads to severe developmental defects. Therefore, it is crucial to understand the mechanisms initiating and controlling polar proteins localisation. Next to polarly distributed PIN's, there is a growing group of polarly localized proteins transporting hormones or nutrients placed at the outer lateral and inner lateral polar domains. In my work I was mostly focused on polarity and function of auxinic-like compounds transporter ABCG37/PIS1, which localises to outer lateral domain in epidermal cells. I tried to characterise the transporting function of this specifically localised protein and find the regulators and mechanisms determining polarity. In order to get a more global overview about components and processes controlling asymmetric distribution of proteins I have included other asymmetrically distributed proteins like ABCG36, BOR4 or BOR1 localised to outer- or inner-lateral domains, respectively

Unraveling the role of Arabidopsis ALIX in the trafficking and turnover of abscisic acid receptors

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-11-2019Esta tesis tiene embargado el acceso al texto completo hasta el 22-05-2021The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FYVE1) and VACUOLAR PROTEIN SORTING 23A (VPS23A), components of the endosomal sorting complex required for transport (ESCRT)-I complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. Here we show that Arabidopsis thaliana ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors.El tráfico endosomal permite a las plantas controlar la abundancia de proteínas transmembrana y proteínas solubles asociadas transitoriamente a ella como ocurre con los receptores de ácido abscísico de la familia PYR/PYL/RCAR (PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS). La degradación vacuolar de estos receptores ocurre a través de la ruta endosomal y está mediada por FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FYVE1) y VACUOLAR PROTEIN SORTING 23A (VPS23A), dos proteínas pertenecientes al complejo ESCRT-I (endosomal sorting complex required for transport). Tanto FYVE1 como VPS23A son capaces de interaccionar con ALIX (ALG-2 INTERACTING PROTEIN-X), una proteína asociada al complejo ESCRT-III. Sin embargo, la relevancia funcional de dichas interacciones y su implicación en la selección y tráfico de proteínas cargo se desconoce. En este trabajo mostramos cómo la proteína ALIX de Arabidopsis thaliana es capaz de interaccionar directamente con los receptors de ABA en endosomas tardíos, provomiendo su degradación. De hecho, alteraciones en la función de ALIX conducen a una acumulación de los receptores de ABA y a una localización subcelular alterada de los mismos. En línea con esto, los mutantes de pérdida de función alix-1, muestran hipersensibilidad al ABA durante el desarrollo y en el cierre estomático, revelando un nuevo papel de la maquinaria ESCRT en el control de la pérdida de agua a través de los estomas. Estas respuestas de hipersensibilidad que presentan los mutantes alix-1 son suprimidas cuando se elimina parcialmente la actividad de los receptores de ABA, lo cuál apoya la idea de que ALIX afecta las respuestas al ABA controlando principalmente la estabilidad de sus receptores. La proteína mutante ALIX-1 muestra una menor interacción con VPS23A y con los receptores de ABA, proporcionando una base molecular que explica la hipersensibilidad al ABA observada en los mutantes alix-1. Nuestro estudio desvela por tanto un nuevo mecanismo de regulación negativa de la ruta del ABA, que actuando a través de la proteína ALIX para controlar la acumulación de PYR/PYL/RCAR específicos

Membrane Traffic and Fusion at Post-Golgi Compartments

Complete sequencing of the Arabidopsis genome a decade ago has facilitated the functional analysis of various biological processes including membrane traffic by which many proteins are delivered to their sites of action and turnover. In particular, membrane traffic between post-Golgi compartments plays an important role in cell signaling, taking care of receptor–ligand interaction and inactivation, which requires secretion, endocytosis, and recycling or targeting to the vacuole for degradation. Here, we discuss recent studies that address the identity of post-Golgi compartments, the machinery involved in traffic and fusion or functionally characterized cargo proteins that are delivered to or pass through post-Golgi compartments. We also provide an outlook on future challenges in this area of research

Plant transporters involved in combating boron toxicity: beyond 3D structures

Version of Record published: 11 August 2020Membrane transporters control the movement and distribution of solutes, including the disposal or compartmentation of toxic substances that accumulate in plants under adverse environmental conditions. In this minireview, in the light of the approaching 100th anniversary of unveiling the significance of boron to plants (K. Warington, 1923; Ann. Bot.37, 629) we discuss the current state of the knowledge on boron transport systems that plants utilise to combat boron toxicity. These transport proteins include: (i) nodulin-26-like intrinsic protein-types of aquaporins, and (ii) anionic efflux (borate) solute carriers. We describe the recent progress made on the structure–function relationships of these transport proteins and point out that this progress is integral to quantitative considerations of the transporter's roles in tissue boron homeostasis. Newly acquired knowledge at the molecular level has informed on the transport mechanics and conformational states of boron transport systems that can explain their impact on cell biology and whole plant physiology. We expect that this information will form the basis for engineering transporters with optimised features to alleviate boron toxicity tolerance in plants exposed to suboptimal soil conditions for sustained food production.Maria Hrmova, Matthew Gilliham and Stephen D. Tyerma

Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity

Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants

Endocytic regulation of alkali metal transport proteins in mammals, yeast and plants

The relative concentrations of ions and solutes inside cells are actively maintained by several classes of transport proteins, in many cases against their concentration gradient. These transport processes, which consume a large portion of cellular energy, must be constantly regulated. Many structurally distinct families of channels, carriers, and pumps have been characterized in considerable detail during the past decades and defects in the function of some of these proteins have been linked to a growing list of human diseases. The dynamic regulation of the transport proteins present at the cell surface is vital for both normal cellular function and for the successful adaptation to changing environments. The composition of proteins present at the cell surface is controlled on both the transcriptional and post-translational level. Post-translational regulation involves highly conserved mechanisms of phosphorylation- and ubiquitylation-dependent signal transduction routes used to modify the cohort of receptors and transport proteins present under any given circumstances. In this review, we will summarize what is currently known about one facet of this regulatory process: the endocytic regulation of alkali metal transport proteins. The physiological relevance, major contributors, parallels and missing pieces of the puzzle in mammals, yeast and plants will be discussed.This work was supported by grant BFU2011-30197-C03-03 from the Ministerio de Ciencia e Innovacion (Spain). V.L.-T. is supported by a fellowship from the Universidad Politecnica de Valencia. C. P. is supported by a fellowship from the Consejo Superior de Investigaciones Cientificas (Spain).Mulet Salort, JM.; Llopis Torregrosa, V.; Primo Planta, C.; Marques Romero, MC.; Yenush, L. (2013). 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