In vitro reconstitution of COPII vesicles from Arabidopsis thaliana suspension-cultured cells

Baiying Li, Yonglun Zeng, Sze Wan Lo, Yusong Guo, Liwen Jiang*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)810-830
Number of pages21
JournalNature Protocols
Volume18
Issue number3
Early online date4 Jan 2023
DOIs
Publication statusPublished - Mar 2023

Scopus Subject Areas

  • Biochemistry, Genetics and Molecular Biology(all)

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