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

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

3. "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 -
"Golgi apparatus (borderless version)-en" by Kelvinsong - Own work. Licensed under CC BY 3.0 via Commons -

4. 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

5. 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)

6. 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 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)

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

8. 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

9. 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

10. 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

11. 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

12. 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 -
Schematic Retromer
Retromer
Pentameric complex
9 subunits
Sorts transmembrane proteins back from the TGN
Vacuolar Sorting Receptors (VSRs)
Auxin efflux carrier (PIN family)

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

14. Thank you for your attention!

15. 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:
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:
Šamaj J., Read N.D., Volkmann D., Menzel D., Baluška F.: The endocytic network in plants. TRENDS in Cell Biology (2005). Vol15 No.,
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:
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:
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: