Proteomics analysis of endosomal compartments in Arabidopsis (ERA-PG 069)
Zusammenfassung der Projektergebnisse
At the start of this project, the endosomal compartments in which plant plasma-membrane (PM) proteins are sorted for degradation in the vacuole or recycling to the PM were morphologically and functionally ill-defined, and their composition in terms of resident and cargo proteins was essentially unknown. The aim of the PRECIAR project was to identify and characterise endosomal proteins of the flowering plant Arabidopsis thaliana. Endosomal membranes were immuno-isolated from transgenic seedlings expressing human transferrin receptor (hTfR) or the a1 subunit of vacuolar ATPase fused to GFP and subjected to MS / LOPIT analysis, yielding a list of candidate endosomal proteins. The immuno-isolation and LOPIT procedures were optimised. Several proteins predicted to be involved in endosomal traffic were characterised by subcellular localisation and functional analysis. These included a collection of protein markers for endocytic compartments, regulatory proteins such as muadaptin, ARF-GEF and SM protein, the phosphorylation pattern of the SERK family of receptors and the subcellular location of the BRI1 receptor. A major emphasis was on the functional characterization of the endosomal compartments. This included the localization of retromer and analysis of its role in the recycling of vacuolar sorting receptors (VSRs). Contrary to previous findings, the results clearly place the sorting nexins (two of the five retromer subunits) to the trans Golgi network (TGN) which in plants acts as the early endosome (EE). Furthermore, post-TGN traffic of vacuolar cargo was demonstrated to occur in a VSR-independent manner, suggesting that VSRs are recycled to a site upstream of the TGN. The expression of endoplasmic reticulum (ER)-anchored lumenal domains of VSRs led to the accumulation of vacuolar cargo in the ER, suggesting that VSR-cargo interactions may start in this compartment. Other work dealt with the dynamics of the TGN, establishing that both endocytic and secretory cargo pass through the TGN, and that the TGN can be released from and reassociate with the Golgi stack. Further work has shown that multivesicular bodies (the late endosome, LE, of plants) develop out of the TGN through a process of maturation. Another aim was to study the role of ubiquitination in endocytosis of membrane proteins. We designed a strategy to biochemically isolate membrane proteins subjected to this type of regulation. The approach was based on expression in plant cells of tagged ubiquitin, followed by affinity purification of ubiquitinated membrane proteins. This blind-search was unsuccessful, due to insufficient enrichment of the purified proteins. We did not have the resources to attempt improvement of this strategy, so we tried an alternative targeted-search. In this way we have identified three putative ubiquitinated membrane proteins, which we are now characterizing.
Projektbezogene Publikationen (Auswahl)
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(2008) A proteomics approach to membrane trafficking. Plant Physiol. 147(4):1584-9
Groen AJ, de Vries SC, Lilley KS
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(2008) Identification of in vitro phosphorylation sites in the Arabidopsis thaliana Somatic Embryogenesis Receptor-like Kinase family. Proteomics 8, 368-379
Karlova, R., Boeren, S., Kwaaitaal, M., Aker, J., Vervoort, J., de Vries, S.C.
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(2008) peer-reviewed article P2 Sub-cellular localization of membrane proteins. Proteomics 8(19):3991-4011
Sadowski PG, Groen AJ, Dupree P, Lilley KS
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(2009) Post-Golgi traffic in peer-reviewed article P1 plants. Traffic 10(7):819-28
Richter S, Voss U, Jürgens G
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(2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J. 59(1):169-78
Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J
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(2010) Endocytic and secretory traffic in Arabidopsis merge in the TGN/EE, an independent organelle that undergoes reversible homotypic fusion. Plant Cell 22(4):1344-57
Viotti, C., Bubeck, J., Stierhof, Y.-D., Krebs, M., Langhans, M., van den Berg, W., van Dongen, W., Richter, S., Geldner, N., Takano, J., Jürgens, G., de Vries, S.C., Robinson, D.G., Schumacher, K.
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(2010) Improved sub-cellular resolution via simultaneous analysis of organelle proteomics data across varied experimental conditions. Proteomics 10(23):4213-9
Trotter MW, Sadowski PG, Dunkley TP, Groen AJ, Lilley KS
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(2010) Proteomics of total membranes and subcellular membranes. Expert Rev Proteomics 7(6):867-78
Groen AJ, Lilley KS
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(2010) Retromer recycles vacuolar sorting receptors from the trans-Golgi network. Plant J. 61: 107-121
Niemes S, Langhans M, Viotti C, Scheuring D, Yan MSW, Jiang L, Hillmer S, Robinson DG, Pimpl P
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(2010) Sorting of plant vacuolar proteins is initiated in the ER. Plant J. 62: 601-614
Niemes S, Labs M, Scheuring D, Krueger F, Langhans M, Jesenofsky B, Pimpl P
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(2010) Strategies to improve the peer-reviewed article antigenicity, ultrastructure preservation and visibility of trafficking compartments in Arabidopsis tissue. Eur J Cell Biol. 89(2-3):285-97
Stierhof YD, El Kasmi F
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(2010). Curr. Topics Dev. Biol. 91, 1-27
Llavata Peris, C.I., Rademacher, E.H. and Weijers, D.
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(2010). Nature 464, 913-916
Schlereth, A.S., Möller, B., Liu, W., Flipse, J., Kientz, M., Rademacher, E.H., Schmid, M., Jürgens, G. and Weijers, D.
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(2011) Arabidopsis SNARE protein SEC22 is essential for gametophyte development and maintenance of Golgi-stack integrity. Plant J. 66(2):268-79
El-Kasmi F, Pacher T, Strompen G, Stierhof YD, Müller LM, Koncz C, Mayer U, Jürgens G
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(2011) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23: 3463-3481
Scheuring D, Viotti C, Krüger F, Künzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K
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(2012) peer-reviewed article Sec1/Munc18 protein stabilizes fusion-competent syntaxin for membrane fusion in Arabidopsis cytokinesis. Dev Cell 22(5):989-1000
Park M, Touihri S, Müller I, Mayer U, Jürgens G
