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Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana

Molecular Plant 2015年第8期8卷 1175-1187页

作者： Marta Zwiewka tomasz nodzynski Stephanie Robert Steffen Vanneste Jiri Friml Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology (CEITEC) Masaryk University Kamenice 5 CZ-625 00 BrnoCzech Republic Department of Plant Systems Biology VlB and Department of Plant Biotechnology and Bioinformatics Ghent University Technologiepark 927 9052 Gent Belgium Institute of Science and Technology (IST) Austria Am Campus 1 3400 Klosterneuburg Austria Present address: Department of Forest Genetics and Plant Physiology Swedish University of Agricultural Sciences/Ume~. Plant Science Center 901 83 UmeaSweden

The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- motic conditions disrupt the equilibrium of the plasma membrane： hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root mer- istem cells. Acute hyperosmotic treatments （ionic and nonionic） enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presum- ably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin- mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

关键词： osmotic stress protein trafficking clathrin-mediated endocytosis auxin

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Enquiry into the Topology of Plasma Membrane- Localized PIN Auxin Transport Components

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Molecular Plant 2016年第11期9卷 1504-1519页

作者： tomasz nodzynski Steffen Vanneste Marta Zwiewka Marketa Pernisova Jan Hejatko Jiri Friml CEITEC - Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic Department of Plant Systems Biology VIB 9052 Gent Belgium Department of Plant Biotechnology and Bioinformatics Ghent University 9052 Gent Belgium National Centre for Biomolecular Research Masaryk University Kamenice 5 62500 Brno Czech Republic Institute of Science and Technology Austria (IST Austria) Am Campus 1 3400 Klosterneuburg Austria

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regard- less of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immuno- localization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane （TM） bundles of five m-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

关键词： plasma membrane protein topology auxin efflux carriers Arabidopsis thaliana

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Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment （PVC） Function in Arabidopsis

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Molecular Plant 2013年第6期6卷 1849-1862页

作者： tomasz nodzynski Mugurel I. Ferarub Sibylle Hirsch Riet De Rycke Claudiu Niculaes Wout Boerjan Jelle Van Leene Geert De Jaeger Steffen Vanneste Jiri Friml Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology (CEITEC) Masaryk University (MU) Kamenice 5 CZ-625 00Brno Czech Republic Department of Plant Systems Biology VIB and Department of Plant Biotechnology and Bioinformatics Ghent University 9052 Gent Belgium Institute of Science and Technology Austria (IST Austria) Am Campus 1 3400 Klosterneuburg Austria Present address: Department of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences (BOKU) 1190 Vienna Austria Present address: Institute of Plant Biology University of Zurich 8008 ZCirich Switzerland

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 （pat3） mutant that we identified by an epifluorescence-based for- ward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFR While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compart- ments （PVC） with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A （VPS35A）, a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lyric vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.

关键词： retromer VPS35 VPS29 prevacuolar compartment (PVC) vacuolar trafficking Arabidopsis thaliana.

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