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      Golgi-localized LOT regulates trans-Golgi network biogenesis and pollen tube growth

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      Proceedings of the National Academy of Sciences
      Proceedings of the National Academy of Sciences

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          Abstract

          The trans-Golgi network (TGN) is an essential tubular-vesicular organelle derived from the Golgi and functions as an independent sorting and trafficking hub within the cell. However, the molecular regulation of TGN biogenesis remains enigmatic. Here we identified an Arabidopsis mutant loss of TGN ( lot) that is defective in TGN formation and sterile due to impaired pollen tube growth in the style. The mutation leads to overstacking of the Golgi cisternae and significant reduction in the number of TGNs and vesicles surrounding the Golgi in pollen, which is corroborated by the dispersed cytosolic distribution of TGN-localized proteins. Consistently, deposition of extracellular pectin and plasma membrane localization of kinases and phosphoinositide species are also impaired. Subcellular localization analysis suggests that LOT is localized on the periphery of the Golgi cisternae, but the mutation does not affect the localization of Golgi-resident proteins. Furthermore, the yeast complementation result suggests that LOT could functionally act as a component of the guanine nucleotide exchange factor (GEF) complex of small Rab GTPase Ypt6. Taken together, these findings suggest that LOT is a critical player for TGN biogenesis in the plant lineage.

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          Pectin methylesterases: cell wall enzymes with important roles in plant physiology.

          Pectin methylesterases catalyse the demethylesterification of cell wall polygalacturonans. In dicot plants, these ubiquitous cell wall enzymes are involved in important developmental processes including cellular adhesion and stem elongation. Here, I highlight recent studies that challenge the accepted views of the mechanism and function of pectin methylesterases, including the co-secretion of pectins and pectin methylesterases into the apoplasm, new action patterns of mature pectin methylesterases and a possible function of the pro regions of pectin methylesterases as intramolecular chaperones.
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            Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis.

            In eukaryotic cells, compartments of the highly dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H(+)-ATPases (V-ATPases). In the Arabidopsis thaliana genome, most V-ATPase subunits are encoded by small gene families, thus offering potential for a multitude of enzyme complexes with different kinetic properties and localizations. We have determined the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a. Colocalization experiments as well as immunogold labeling showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocks the endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Furthermore, nascent cell plates are rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Together, our results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.
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              Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

              Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.
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                Author and article information

                Journal
                Proceedings of the National Academy of Sciences
                Proc Natl Acad Sci USA
                Proceedings of the National Academy of Sciences
                0027-8424
                1091-6490
                November 27 2018
                November 27 2018
                November 27 2018
                November 09 2018
                : 115
                : 48
                : 12307-12312
                Article
                10.1073/pnas.1809206115
                6275481
                30413616
                65bfdf84-1206-48d3-845e-f1b83bae50a3
                © 2018

                Free to read

                http://www.pnas.org/site/misc/userlicense.xhtml

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