The structure of the COPI coat determined within the cell.

Yury S Bykov,Miroslava Schaffer,John Ag Briggs,Jürgen M Plitzko,Svetlana O Dodonova,Benjamin D Engel,Wolfgang Baumeister,Sahradha Albert

doi:10.7554/elife.32493

Yury S Bykov, Miroslava Schaffer + Show 6 more

Open Access

https://doi.org/10.7554/elife.32493

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Abstract

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

Highlights

Vesicular transport between cellular compartments is a fundamental mechanism of eukaryotic cells
Similar architecture is found in the clathrin heavy chain, the Sec31 COPII coat subunits, and some nuclear pore components (Lee and Goldberg, 2010; Devos et al, 2004)
All three archetypal protein coats could be visually identified in the tomograms without any ambiguity: clathrin-coated vesicles were found in the vicinity of the TGN and were distinguished by their characteristic triskelion-based cage (Figure 1C,C’), while ER exit sites with COPII buds and vesicles were distinguished by their two-layered coat (Figure 1D,D’)

Summary

Introduction

Vesicular transport between cellular compartments is a fundamental mechanism of eukaryotic cells. Archetypal protein coats, COPI, COPII and clathrin, mediate the formation and trafficking of vesicles in the endocytic-secretory pathway (Bonifacino and Glick, 2004). These coats share organizational and functional principles, and likely diverged from an ancestral coat prior to the last common eukaryotic ancestor. We previously used cryo-electron tomography and subtomogram averaging (Wan and Briggs, 2016) to build a molecular model of the COPI coat budded in vitro from giant unilamellar vesicles using purified coat protein components (Faini et al, 2012; Dodonova et al, 2015, Dodonova et al, 2017). In situ cryo-ET studies of this organism revealed the presence of ordered intracisternal arrays that may help maintain the architecture of the C. reinhardtii Golgi (Engel et al, 2015)

Results and discussion

Materials and methods

Funding Funder Deutsche Forschungsgemeinschaft