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. 2019 Jan 9:9:1972.
doi: 10.3389/fpls.2018.01972. eCollection 2018.

Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings

Niccolò Mosesso  1 , Tobias Bläske  1 , Marie-Kristin Nagel  1 , Michael Laumann  2 , Erika Isono  1
Affiliations

Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings

Niccolò Mosesso et al. Front Plant Sci. .

Abstract

Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs. However, many of the details during plant CCV formation are not completely elucidated. The analysis of isolated CCVs is therefore important to better understand the formation of plant CCVs, their cargos and the regulation of clathrin-mediated transport processes. In this article, we describe an optimized method to isolate CCVs from Arabidopsis thaliana seedlings.

Keywords: Arabidopsis thaliana; clathrin coated vesicles; density fractionation; negative staining; scanning electron microscopy.

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Figures

FIGURE 1
FIGURE 1
Schematic representation of the CCV-isolation procedure.
FIGURE 2
FIGURE 2
Negative STEM micrograph of the final CCV fraction prepared from 7-day-old wild-type seedlings. Scale bar: 200 nm.
FIGURE 3
FIGURE 3
CCVs were prepared from total plant extract of 7-day-old wild type (A, B) mRFP-ARA7 seedlings. Samples were collected during the procedure of CCV isolation and subjected to Western blot analyses using antibodies against CHC, mRFP, and various subcellular organelle marker proteins. CCV, CCV-containing fraction; DFGL, linear D2O/Ficoll gradient load; S1, supernatant after 1,000 ×g centrifugation; S30, supernatant after 30,000 ×g centrifugation; SGL, sucrose step gradient load. The following antibodies were used as organelle- or compartment-specific markers: anti-V-ATPase (vacuole), anti-TOC75 (chloroplast), anti-BiP2 (ER), anti-Sec21p (COPI).
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