Abstract
The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.
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Acknowledgements
We thank Drs Chris Somerville (UC Berkeley, USA), Niko Geldner (University of Lausanne, Switzerland), and Herman Höfte (INRA, France) for providing the transgenic Arabidopsis lines CESA6-YFP, RABD2b-YFP (Wave 33), and CESA3-GFP, respectively. We also thank Drs Gerd Jürgens (University of Tuebingen, Germany), Paul Schulze-Lefert (Max Planck Institute for Plant Breeding Research, Germany), Richard Williamson (Australian National University) for providing SNAP33, PEN1 (SYP121) and SYP72 and CESA1 antibodies. We thank Drs Thomas Girke (UC Riverside, USA), Brett S Phinney (UC Davis, USA) and Mark Gjukich (University of Michigan, USA) for their support on data deposition. This work was funded by a grant from the DOE (DEFG03-02ER15295) to NVR. GD was supported by UC Davis startup funds.
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Supplementary information
Supplementary information, Figure S1
Fractionation of SYP61 vesicles. (PDF 45 kb)
Supplementary information, Figure S2
LC/MS quantitative analysis using MS spectra intensity derived from extracted ion chromatograms (XIC). (PDF 275 kb)
Supplementary information, Figure S3
SYP61 colocalizes with isolated components of SNARE complexes VPS45, SYP43, and the GTPase RABD2B. (PDF 123 kb)
Supplementary information, Figure S4
SYP61 colocalization with VSR2. (PDF 47 kb)
Supplementary information, Figure S5
Colocalization of SYP61 with CESA1 in Arabidopsis protoplasts. (PDF 45 kb)
Supplementary information, Figure S6
Colocalization of SYP61 with SCAMP. (PDF 84 kb)
Supplementary information, Table S1
Proteins detected in only SYP61 but not IgG (XLS 218 kb)
Supplementary information, Table S2
Proteins detected in both SYP61 and IgG fractions with 1% FDR (XLS 49 kb)
Supplementary information, Table S3
Primers used for cDNA amplifications (PDF 76 kb)
Supplementary information, Data S1
Materials and Methods (PDF 7 kb)
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Drakakaki, G., van de Ven, W., Pan, S. et al. Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis. Cell Res 22, 413–424 (2012). https://doi.org/10.1038/cr.2011.129
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DOI: https://doi.org/10.1038/cr.2011.129
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