In vitro COPII Reconstitution Reveals Novel Insight of Arabidopsis ER Export
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摘要The molecular mechanisms of COPII vesicle formation and function have been well illustrated via in vitro reconstitution system in both yeast and mammalian models [1, 2]. However, COPII-mediated ER-to-Golgi protein trafficking in plant is more complicated due to the existence of two models: the COPII vesicles and the ER-Golgi tubular connections [3]. More interestingly, plant COPII paralogs outnumber those in other eukaryotes. The Arabidopsis thaliana genome contains five Sar1 homologs (AtSar1a/b/c/d/x), with their functional diversity previously unknown. Recently it has been demonstrated that a single amino acid difference in AtSar1a is pivotal for its specific cellular localization and the formation of the unique AtSar1a/AtSec23a pair, which contributes to the distinct function of AtSar1a/AtSec23a on cargo trafficking in plants, especially ER stress responses [4, 5]. Here we have established an in vitro COPII vesicle reconstitution system using plant-derived materials and used this system to further identify cargo specificity of individual AtSar1 paralogs and their functional partners. By incubating Arabidopsis-derived microsome-enriched fractions and pre-cleared cytosol with the energy regeneration systems, we first reconstituted plant COPII vesicles in vitro. Reconstituted vesicles were purified by OptiPrep floatation assay for morphological study using TEM analysis. The concentrated vesicles with a diameter of 70-100 nm were identified by immunogold labelling with Sec22 antibodies, which is in consistence with COPII vesicles in yeast and mammal. In addition, the vesicle production was shown to be AtSar1-dependent and could be inhibited by adding GTPrs, the dominant negative form of AtSar1 recombinant proteins or Anti-Sar1 serum, proving its COPII identity. More interestingly, LC-MS/MS analysis of in vitro reconstituted COPII vesicles further expands the existing knowledge of COPII cargoes, which includes families of transporters and channels without conventional signal peptides. This newly established in vitro COPII reconstitution system not only provides evidence for the plant ER-to-Golgi COPII vesicle trafficking, but also serves as a novel and powerful tool to further study the aforementioned functional diversity of plant COPII homologs in vesicle formation and ER cargo selection in plants. Supported by grant from the Research Grants Council of Hong Kong (CUHK14130716, CUHK2/CRF/11G, C4011-14R, C4012-16E and AoE/M-05/12).
著者Baiying LI, Yonglun ZENG, Xiangfeng WANG, Yusong GUO, Liwen JIANG
會議名稱Cold Spring Harbor Asia conferences on Plant Cell and Development Biology

上次更新時間 2018-16-04 於 18:37