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Identification of regulatory and cargo proteins of endosomal and secretory pathways in Arabidopsis thaliana by proteomic dissection
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Heard, William, Sklenar, Jan, Tomé, Daniel F. A., Robatzek, Silke and Jones, Alexandra M. (2015) Identification of regulatory and cargo proteins of endosomal and secretory pathways in Arabidopsis thaliana by proteomic dissection. Molecular & Cellular Proteomics, 14 (7). pp. 1796-1813. doi:10.1074/mcp.M115.050286
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Official URL: http://dx.doi.org/10.1074/mcp.M115.050286
Abstract
The cell s endomembranes comprise an intricate, highly dynamic and well-organised system. In plants the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi Network (TGN), Early Endosomes (EE), secretory vesicles, Late Endosomes (LE), Multivesicular Bodies (MVB) and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localises to the Golgi, Clathrin Light Chain 2 (CLC2) labelling Clathrin-coated vesicles and pits and the Vesicle Associated Membrane Protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNARES and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA and recently defined complexes such as TPLATE. The sub-cellular localisation of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic data set to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localisation of proteins, identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.
| Item Type: | Journal Article | ||||||||||
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| Subjects: | Q Science > QH Natural history | ||||||||||
| Divisions: | Faculty of Science > Life Sciences (2010- ) | ||||||||||
| Library of Congress Subject Headings (LCSH): | Coated vesicles, Arabidopsis thaliana, Plant proteomics | ||||||||||
| Journal or Publication Title: | Molecular & Cellular Proteomics | ||||||||||
| Publisher: | American Society for Biochemistry and Molecular Biology | ||||||||||
| ISSN: | 1535-9476 | ||||||||||
| Official Date: | 1 July 2015 | ||||||||||
| Dates: |
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| Volume: | 14 | ||||||||||
| Number: | 7 | ||||||||||
| Number of Pages: | 18 | ||||||||||
| Page Range: | pp. 1796-1813 | ||||||||||
| DOI: | 10.1074/mcp.M115.050286 | ||||||||||
| Status: | Peer Reviewed | ||||||||||
| Publication Status: | Published | ||||||||||
| Access rights to Published version: | Restricted or Subscription Access | ||||||||||
| Funder: | Gatsby Charitable Foundation (GCF) | ||||||||||
| Adapted As: |
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