Fujiwara M, Uemura T, Ebine K, Nishimori Y, Ueda T, Nakano A, Sato MH, Fukao Y

Membrane trafficking in plants is involved in cellular development and the adaptation to various environmental changes. SNARE proteins mediate the fusion between vesicles and organelles to facilitate transport cargo proteins in cells. To further characterize SNARE protein networks in cells, we carried out interactome analysis of SNARE proteins using 12 ...

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transgenic Arabidopsis thaliana plants expressing GFP-tagged Qa-SNAREs (SYP111, SYP121, SYP122, SYP123, SYP132, SYP21, SYP22, SYP31, SYP32, SYP41, SYP42 and SYP43). Microsomal fractions were prepared from each transgenic root, and subjected to immunoprecipitation (IP) using micro-magnetic beads coupled to anti-GFP antibodies. To identify Qa-SNARE-interacting proteins, all IP products were then subjected to mass spectrometric (IP-MS) analysis. The IP-MS data revealed not only known interactions of SNARE proteins, but also unknown interactions. The IP-MS results were next categorized by gene ontology analysis. Data revealed that categories of cellular component organization, cytoskeleton and endosome were enriched in the SYP2, SYP3 and SYP4 groups. In contrast, transporter activity was classified specifically in the SYP132 group. We also identified a novel interaction between SYP22 and VAMP711, which was validated using co-localization analysis with confocal microscopy and immunoprecipitation. Additional novel SNARE-interacting proteins play roles in vesicle transport and lignin biosynthesis, and were identified as membrane microdomain-related proteins. We propose that Qa-SNARE interactomics is useful for understanding SNARE interactions across the whole cell.

Date: Feb. 19, 2014

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