A structure of the COPI coat and the role of coat proteins in membrane vesicle assembly

Abstract

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.

Structural basis for the binding of tryptophan-based motifs by δ-COP

“…The hyper-open conformation of coatomer, in which δ-COP MHD is on the very outside of the coat away from the membrane, would be maintained in the vesicle coat as long as coatomer was bound to Arf1:GTP. After nucleotide hydrolysis and Arf1:GDP disengagement, the F-subcomplex likely would revert to a more closed form; however, because of the COPI B-subcomplex binding to KKxx and KxKxx transmembrane cargoes (29)(30)(31), the coat should not dissociate from the vesicle surface immediately (5). Assuming that the F-subcomplex adopts a closed conformation similar to that of a cytosolic AP complex, modeling suggests that the Wx n (1)(2)(3)(4)(5)(6) [WF] binding site on δ-COP should be accessible when the F-subcomplex is closed (Fig.…”

“…However, the open form of F-subcomplexes attached to a membrane in a coat recently has been demonstrated to be even more extreme than the open conformation of AP complexes (27,28) and now is referred to as "hyper-open" (29). The hyper-open conformation of coatomer, in which δ-COP MHD is on the very outside of the coat away from the membrane, would be maintained in the vesicle coat as long as coatomer was bound to Arf1:GTP.…”

“…Therefore one intriguing possibility is that the high global negative charge of δ-COP may contribute to the specificity of target membrane fusion of COPI-coated vesicles that in δ-COP could inhibit close apposition and hence fusion with more negatively charged membranes such as the late Golgi/TGN, but not inhibit contact with the comparatively uncharged membranes of the cis-Golgi and ER. Such a mechanism could impart retrograde directionality to COPI vesicle transport through the Golgi toward the ER and also might involve the α-COP CTD /e-COP subcomplex, which likewise is both highly negatively charged (36,37) and located on the outside of the COPI coat (29). It should be noted that for such a scenario to operate, a significant proportion of coatomer must indeed remain associated with vesicles right up to their docking step to allow Dsl1 complex-mediated tethering of COPI-coated vesicles to occur (5,15).…”

Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway

“…ER-derived cargo is first shuttled to the ERGIC in a coat protein (COP) Ⅱ-dependent step and subsequently to the Golgi apparatus in a second vesicular transport step involving COPⅠ-coated vesicles, RAB and ARF GTPases, as well as cytoskeletal networks; incoming vesicles can also be recycled to the ER in a COPⅠ-mediated process [28] . The ERGIC contributes to the concentration, folding, and quality control of newly synthesized proteins and is required for the production of several viral pathogens [29] .…”

The Nobel Prize in Chemistry 2017: High-Resolution Cryo-Electron Microscopy

“…To improve the signals and resolution, repetitive molecular features in a 3D tomogram can be further extracted computationally and subjected to averaging and classification using methods similar to those employed in single particle analysis (Asano et al, 2016;Beck & Baumeister, 2016). These methods, called subtomogram averaging, provided valuable insights into biological phenomena such as HIV maturation (Mattei et al, 2016;Schur et al, 2014Schur et al, , 2016, nuclear pore structures (Beck & Glavy, 2014;Beck et al, 2007;Bui et al, 2013), coat protein complex I (Dodonova et al, 2015(Dodonova et al, , 2017, and ribosometranslocation machinery (Pfeffer et al, 2015). However, a major drawback of these Cryo-electron tomography and subtomogram averaging technique is their limited resolution.…”