Transport vesicles mediate protein traffic between endomembrane organelles in a highly selective and efficient manner. In vitro reconstitution systems have been widely used for studying mechanisms of vesicle formation, polar trafficking, and cargo specificity in mammals and yeast. However, this technique has not yet been applied to plants because of the large lytic vacuoles and rigid cell walls. Here, we describe an Arabidopsis-derived in vitro vesicle formation system to reconstitute, purify and characterize plant-derived coat protein complex II (COPII) vesicles. In this protocol, we provide a detailed method for the isolation of microsomes and cytosol from Arabidopsis thaliana suspension-cultured cells (7–8 h), in vitro COPII vesicle reconstitution and purification (4–5 h) and biochemical and microscopic analysis using specific antibodies against COPII cargo molecules for reconstitution efficiency evaluation (2 h). We also include detailed sample-preparation steps for analyzing vesicle morphology by cryogenic electron microscopy (1 h) and vesicle cargoes by quantitative proteomics (4 h). Routinely, the whole procedure takes ~18–20 h of operation time and enables plant researchers without specific expertise to achieve organelle purification or vesicle reconstitution for further characterization.
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The authors thank R. Schekman, A. Gorur and D. G. Robinson for critical suggestions; D. Inzé for the PSB-D suspension cultures; X. Wang for sharing experiences on fractionation and cytosol preparation; and Y. Chen for microscopic images. This work was supported by grants from the National Natural Science Foundation of China (91854201), the Research Grants Council of Hong Kong (CUHK14100818, 14101219, C4033-19E, C4002-17G, C4002-20W, C4002-21EF, C2009-19G, C4041-18E, R4005-18 and AoE/M-05/12), the CUHK Research Committee and CAS-Croucher Funding Scheme for Joint Laboratories to L.J.
The authors declare no competing interests.
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Nature Protocols thanks Fernando Aniento, Liang Ge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol:
Li, B. et al. Nat. Plants 7, 1335–1346 (2021): https://doi.org/10.1038/s41477-021-00997-9
Source Data Fig. 1
Uncropped confocal images for Fig. 1c
Source Data Fig. 2
Uncropped confocal images for Fig. 2b
Source Data Fig. 3
Uncropped TEM image for Fig. 3c
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Li, B., Zeng, Y., Lo, S.W. et al. In vitro reconstitution of COPII vesicles from Arabidopsis thaliana suspension-cultured cells. Nat Protoc 18, 810–830 (2023). https://doi.org/10.1038/s41596-022-00781-9
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