Endocytic network in plants trans - Golgi network Oskar Liebisch Wednesday, 4th of November, 2015

Structure Where is the trans – Golgi network located? Plant endosomal trafficking (EE, LE) Signaling proteins

"Plant cell structure-en" by LadyofHats - Self-made using Adobe Illustrator. (The original edited was also made by me, LadyofHats). Licensed under Public Domain via Commons - https://commons.wikimedia.org/wiki/File:Plant_cell_structure-en.svg#/media/File:Plant_cell_structure-en.svg "Golgi apparatus (borderless version)-en" by Kelvinsong - Own work. Licensed under CC BY 3.0 via Commons - https://commons.wikimedia.org/wiki/File:Golgi_apparatus_(borderless_version)-en.svg#/media/File:Golgi_apparatus_(borderless_version)-en.svg http://cytochemistry.net/_Media/golgi1a_med_hr.jpeg

TGN hypothesis Toyooka et al. 2009: Viotti et al. 2010: TGN fuses with the PM releasing its cargo to the extracellulare space  completely fuse with PM Viotti et al. 2010: TGN cycles between Golgi associated and free states Kang et al. 2011: Short living structure, that releases secretory and clathrin coated vesicles, gives rise to the MVB by their remnant membranes

Kang et al. (2011) I. Golgi trans-type compartment; transformation is coupled by the recruitment of RabA4b and its effector protein PI-4Kß1 (identification as TGN) II. transformation of the trans-most Golgi cisterna into a TGN; accompanied by 3 structural changes: Onset of separation of trans-most cisterna 30-35% reduction of the cisternal membrane area Formation of round SV buds, which are not confined to the trans-Golgi cisterna Found in Kang et al. (2011) Found in Kang et al. (2011)

The trans – Golgi network VHA-a1 subunit of V-ATPase marked red with Red Fluorescent Protein; found in Viotti et al. (2010) At first it was known as a subcompartment of the Golgi BUT Newest study indicating TGN to be a free, highly mobile organelle Early Endosome Matures from the Golgi; study of Viotti et al. 2010 indicate that it can reassociate with V-ATPase required for identity of TGN? Characterized by associated proteins like RabA4b, PI-4k, Rab GTPase, SNARE proteins, VHA-a1 (subunit of V-ATPase) Receives endocytosed cargo from PM (e.g. carbohydrates, proteins) biosynthetic sorting station Forms Clathrin coated vesicles (TGN – MVB, Vacuole), secretory vesicles (TGN – PM) VHA-a1 immunogold labeled in ConcA treated cells; found in Viotti et al. (2010)

Plant endosomal trafficking pathways; found in Reyes et al. (2011)

Early endosome (EE) / Late Endosome What is an endosome? Compartments of the endocytic membrane transport pathway From the PM to the lysosome From the PM to the TGN Early endosome (EE) / Late Endosome

Plant endosomal trafficking After treatmemt with the V-ATPase inhibitor Concanamycin A TGN associated with the Golgi Number of MVB decreased Early/ recycling endosomes Late endosomes/ Multivesicular bodies (MVB) Tubular-vesicular structure, bearing clathrin coated vesicles (CCV) First branching point for: endocytosed and newly synthezised proteins Receive and recycle of: 1. endocytosed membrane proteins; back to PM or to the MVB 2. Vacuolar cargo receptors back to TGN Prevacuolar compartments (intermediate) Spherical Derived from the TGN Sorting membrane proteins into endosomal intraluminal vesicles (ILVs); degradation by fusion with the tonoplast (degradation by vacuoles/lysosomes) Carrying also new proteins from the Golgi to lysosomes/vacuoles

How does the sorting work? Mediators Example of function ESCRTs (Endosomal Sorting Complex Required for Transport) Transmembrane proteins; degredation Retromer Vacuolar Sorting Receptors (VSRs) Extracellular secretion (PIN protein) Ubiquitin BOR1 degradation ADP-ribosylation factor (ARF) maschinery PIN and AUX1 (GNOM) Coating complexes and mediators

ESCRT-0 recognition of PI3P (Phosphatidylinositol 3-phosphate; phospholipe in the PM, serves as binding station for some sorting proteins) via the FYVE domain Clusters cargo ESCRT-1 + ESCRT-2 induce the formation of buds ESCRT-3 mediates vesicle fission ESCRTs are needed to internalize proteins into the MVB (ILVs), because only the outer membrane of the MVB can fuse with the vacuole and release the ILVs for degradation ESCRTs Mechanism of degradation of a PM bound protein; Found in: David Scheuring (Dissertation 2011), not published In fungi and metazoans: 4 multimeric ESCRT related complexes have been identified to be related to the endosomes But not in plants! ESCRT complex Ortholog TOM1 1 ELCH Protein 2 - 3 CHMP1

Retromer Pentameric complex 9 subunits Plant endosomal trafficking pathways; found in Reyes et al. (2011) "Retromer protein complex" by Kianzoidlee - Using Pymol and adobe illustratorPreviously published: Used for my honours thesis. Licensed under CC BY-SA 3.0 via Commons - https://commons.wikimedia.org/wiki/File:Retromer_protein_complex.png#/media/File:Retromer_protein_complex.png Schematic Retromer Retromer Pentameric complex 9 subunits Sorts transmembrane proteins back from the TGN Vacuolar Sorting Receptors (VSRs) Auxin efflux carrier (PIN family)

Example BOR1: Ubiquitination Ubiquination of BOR1; found in Reyes et al. 2011

Thank you for your attention!

References Gendre D., McFarlane H.E., Johnson E., Mouille G., Sjödin A., Oh J., Levesque-Tremblay G., Watanabe Y., Samuels L., Bhaleraoa R.P.: Trans-Golgi Network Localized ECHIDNA/Ypt Interacting Protein Complex Is Required for the Secretion of Cell Wall Polysaccharides in Arabidopsis. The Plant Cell (2013), Vol. 25: 2633–2646. Kang BH, Nielsen E, Preuss ML, Mastronarde D, Staehelin LA: Electron tomography of RabA4b- and PI-4Kbeta1-labeled trans Golgi network compartments in Arabidopsis. Traffic (2011), 12:313-329. Kleine-Vehn J., Dhonukshe P., Swarup R., Bennett M., Frimla J.: Subcellular Trafficking of the Arabidopsis Auxin Influx Carrier AUX1 Uses a Novel Pathway Distinct from PIN1. The Plant Cell (2006), Vol. 18, 3171–3181. Reyes F.C., Buono R. and Otegui M.S.: Plant endosomal trafficking pathways: Current Opinion in Plant Biology (2011), 14: 666-673. Šamaj J., Read N.D., Volkmann D., Menzel D., Baluška F.: The endocytic network in plants. TRENDS in Cell Biology (2005). Vol15 No., 425-433. Scheuring D.: Funktionelle Charakterisierung pflanzlicher Endosomen. (2011); not published. Scheuring D., Viotti C., Krüger F., Künzl F., Sturm S., Bubeck J., Hillmer S., Frigerio L., Robinson D.G., Pimpl P., Schumacher K.: Multivesicular Bodies Mature form the Trans-Golgi Network/Early Endosome in Arabidopsis. The Plant Cell (2011), Vol.23: 3463-3481. Toyooka K, Goto Y, Asatsuma S, KoizumiM, Mitsui T, Matsuoka K: A mobile secretory vesicle cluster involved in mass transport from the Golgi to the plant cell exterior. Plant Cell 2009, 21:1212-1229. Viotti C, Bubeck J, Stierhof YD, Krebs M, Langhans M, van den Berg W, van Dongen W, Richter S, Geldner N, Takano J et al.: Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell (2010), 22:1344-1357.