Role of the Arabidopsis TRAPPII tethering complex in protein sorting at the Trans-Golgi-Network
Final Report Abstract
The trans-Golgi-network (TGN) has essential housekeeping functions in secretion, endocytosis and protein sorting, but also more specialized functions in plant development. How the robustness of basal TGN function is ensured while specialized functions are differentially regulated was poorly understood. Our studies investigate two key regulators of TGN structure and function, ECHIDNA and the Transport Protein Particle II (TRAPPII) tethering complex. An analysis of physical, network and genetic interactions suggest that two network communities are implicated in TGN function and that ECHIDNA and TRAPPII belong to distinct yet overlapping pathways. While ECHIDNA and TRAPPII colocalized at the TGN in interphase cells, their localization diverged in dividing cells. Moreover, ECHIDNA and TRAPPII localization patterns were mutually independent. TGN structure, endocytosis and sorting decisions were differentially impacted in echidna and trappii mutants. Our analyses point to a partitioning of specialized TGN functions, with ECHIDNA being required for cell elongation and TRAPPII for cytokinesis. Two independent pathways able to compensate for each other might contribute to the robustness of TGN housekeeping functions and to the responsiveness and fine tuning of its specialized functions. Our studies have considerably furthered an understanding of TRAPPII’s role in exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, however, little was known about Arabidopsis TRAPPII complex topology and molecular function. This we have addressed, in collaboration with the laboratory of Ian Moore, with convergent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis TRAPPII differs from yeast but resembles metazoan TRAPPII complexes. Cell biological and genetic analyses of Rab-A variants in trappii backgrounds provide additional lines of evidence for the conclusion that the TRAPPII complex behaves as a Guanine-nucleotide Exchange Factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding the control of GEF-Rab interactions will be crucial to unraveling the co-ordination of membrane traffic.
Publications
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2016. The Membrane-Associated Sec1/Munc18 KEULE is Required for Phragmoplast Microtubule Reorganization During Cytokinesis in Arabidopsis. Mol. Plant 9: 528–540
Steiner A, Müller L, Rybak K, Vodermaier V, Facher E, Thellmann M, Ravikumar R, Wanner G, Hauser M-T, and Assaad FF
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2017. Multisubunit tethering complexes in higher plants. Curr Opin. Plant Biol. 40: 97–105. AS 110/4-7
Ravikumar R, Steiner A, Assaad FF
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2017. Plant cytokinesis: Terminology for structures and processes. Trends Plant Sciences. 27(12): 885-894
Smertenko A, Assaad FF, Baluška F, Bezanilla M, Buschmann H, Drakakaki G, Hauser MT, Janson M, Mineyuki Y, Moore I, Müller S, Murata T, Otegui MS, Panteris E, Rasmussen C, Schmit AC, Šamaj J, Samuels L, Staehelin LA, Van Damme D, Wasteneys G, Žárský V
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2018. Independent yet overlapping pathways ensure the robustness and responsiveness of trans-Golgi network functions in Arabidopsis. Development 145(21)
Ravikumar R, Kalbfuß N, Gendre D, Steiner A, Altmann M, Altmann S, Rybak K, Edelmann H, Stephan F, Lampe M, Facher E, Wanner G, Falter-Braun P, Bhalerao RP, Assaad FF
