Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes

Arakel, E.

Schwappach, B.

eLife

English

MPG.PuRe

VESICLESLooking inside the cellAdvances in imaging techniques have shed new light on the structure ofvesicles formed by COPI protein complexes.ERIC C ARAKEL AND BLANCHE SCHWAPPACHVesicles perform a wide range of functionswithin cells, such as the transport of pro-teins and lipids between the differentparts of a cell. Each vesicle is coated with a pro-tein complex, and understanding the structureand function of these complexes is a centralchallenge in cell biology. Two of these com-plexes – COPII and clathrin – are relatively wellunderstood, but the third, which is called COPI,is not.Vesicles coated with COPI are responsible fortransport from the Golgi to the endoplasmicreticulum, and also for transport between thecisternae within the Golgi (Beck et al., 2009).The COPI complex contains seven subunits thatare recruited en bloc to the vesicle membrane(Hara-Kuge et al., 1994). However, the intricatecouplings between these subunits have long hin-dered efforts to determine the molecular archi-tecture of the COPI coats.The basic model of COPI vesicle formationbegins with the recruitment of a GTPase calledArf1 to a membrane. Arf1 is then loaded withGTP, thereby activating it. COPI complexes theninteract with the GTP-loaded Arf1 and cargoproteins to form the vesicle, which can thendetach from the membrane and transport thecargo proteins to their destination. Finally, thecoat disassembles in a process that is called un-coating: three GTPase activating proteins (Arf-GAP1/2/3) play an important role in this processby stimulating the hydrolysis of the GTP and,therefore, deactivating Arf1 (Lanoix et al.,1999; Pepperkok et al., 2000; Weimer et al.,2008). Subsequent studies of this un-coatingprocess suggested that Arf1 and COPI disen-gage from membranes independently, in a non-cooperative manner (Presley et al., 2002;Yang et al., 2002). These and other results ledto a model in which the individual COPI com-plexes were held in place by lateral linkageswithin the coat and by interactions with theunderlying cargo.These studies were built upon in a series ofin-vitro reconstitution experiments undertakenby a collaboration between the groups of FelixWieland (University of Heidelberg) and JohnBriggs (European Molecular Biology Laboratoryand the MRC Laboratory of Molecular Biology).The in-vitro reconstitution approach involvescombining purified proteins and liposomes (atype of vesicle) in a test tube to generate COPI-coated vesicles, thus allowing researchers tostudy the precise sequence of events that takeplace during vesicle formation. Wieland, Briggsand co-workers exploited significant advances intwo techniques – cryo-electron tomography andsubtomogram averaging – to determine themolecular structure of the COPI coat(Dodonova et al., 2017; Dodonova et al.,2015; Faini et al., 2012).However, the process of vesicle formation inthese seminal experiments differed from theprocess in real cells in a number of ways: thevesicles did not contain any cargo, and the Arf1was frozen in its activated state because theresearchers used a variant of GTP called GTPgSthat cannot be hydrolysed. This meant thatCopyright Arakel andSchwappach. This article isdistributed under the terms of theCreative Commons AttributionLicense, which permits unrestricteduse and redistribution provided thatthe original author and source arecredited.Related research article Bykov YS, SchafferM, Dodonova SO, Albert S, Plitzko JM,Baumeister W, Engel BD, Briggs JAG.2017. The structure of the COPI coat deter-mined within the cell. eLife 6:e32493. DOI:10.7554/eLife.32493Arakel and Schwappach. eLife 2017;6:e33650. DOI: https://doi.org/10.7554/eLife.33650 1 of 3INSIGHTseveral questions remained unanswered: Doesearly GTP hydrolysis contribute to a pronouncedconformational change of the coat (such as theexposure of cargo-recognition sites)? Are thelinkages within the coat rearranged to accom-modate cargo? How does Arf1 disengage fromCOPI without perturbing the pre-existing lattice?And how does the cage adapt structurally to theloss of Arf1?The rational next step would have involvedusing GTP (rather than GTPgS), the three Arf-GAPs and cargo proteins in in-vitro reconstitu-tion experiments, which would have been veryarduous. However, in an unexpected develop-ment reported in eLife, Briggs, Benjamin Engeland Wolfgang Baumeister (both of the MaxPlanck Institute of Biochemistry) and co-workers– including Yury Bykov as first author – describethe native structure of COPI-coated vesicles in aspecies of green algae called Chlamydomonasreinhardtii (Bykov et al., 2017). This quantumleap was made possible through a combinationof cryo-focused ion-beam milling (a techniquethat involves vitrifying cells and then using afocused ion beam to slice off ultrathin sections)and further improvements in cryo-electrontomography and subtomogram averaging.Moreover, this approach meant that theresearchers were able to study the structure ofthe coat in the presence of active Arf1, the rele-vant ArfGAPs and the vesicle’s cargo.Building on their previous systematic charac-terization of the morphology of the Golgi in C.reinhardtii (Engel et al., 2015a; Engel et al.,2015b), and what is known about the structuresof COPI, COPII and clathrin, Bykov et al. wereable to identify the three different types ofcoated vesicles in the vicinity of the cisternaewithin the Golgi. The researchers were also ableto determine the in-situ structure of the coatwithout relying on existing structural models,and found that it was remarkably similar to thestructure that emerged from the in-vitro recon-stitution experiments. This finding is significantfor the field because it strongly suggests thatthe existing structural model is physiologicallyrelevant.Moreover, an additional density wasobserved inside the vesicles in C. reinhardtii thatwas not visible in the in-vitro experiments. Thisdensity is likely to correspond to cargo, and thefact that it was observed under the b-propellerof the b’-subunit, but not beneath the b-propel-ler of the a-subunit (which is structurally similar),suggests that this cargo is recognized by a spe-cific subunit of COPI. It is also striking that thisdensity is seen in many vesicles adjoining boththe cis and medial cisternae.Remarkably, the relative proportions of Arf1and COPI were the same for most vesicles, irre-spective of the intactness of the coat: this sug-gests that, upon hydrolysis, the constituents ofthe coat are all released at the same time toallow the vesicle to fuse with an acceptor com-partment, and argues against a step-wise disso-ciation of the coat from the vesicle.In addition to demonstrating the ability of in-situ experiments to distinguish between modelsbased on in-vitro experiments, the work ofBykov et al. highlights the ability of new techni-ques to allow us to see things – such as the den-sity under the b-propeller of the b’-subunit –that we have missed so far. We are keen to geta glimpse of COPII and clathrin in situ too.Eric C Arakel is in the Department of MolecularBiology, University Medical Center Go¨ttingen,Go¨ttingen, GermanyBlanche Schwappach is in the Department ofMolecular Biology, University Medical CenterGo¨ttingen, Go¨ttingen, Germanyblanche.schwappach@med.uni-goettingen.dehttp://orcid.org/0000-0003-0225-6432Competing interests: The authors declare that nocompeting interests exist.Published 05 December 2017ReferencesBeck R, Rawet M, Ravet M, Wieland FT, Cassel D.2009. The COPI system: molecular mechanisms andfunction. FEBS Letters 583:2701–2709. DOI: https://doi.org/10.1016/j.febslet.2009.07.032, PMID: 19631211Bykov YS, Schaffer M, Dodonova SO, Albert S, PlitzkoJM, Baumeister W, Engel BD, Briggs JAG. 2017. Thestructure of the COPI coat determined within the cell.eLife 6:e32493. DOI: https://doi.org/10.7554/eLife.32493, PMID: 29148969Dodonova SO, Diestelkoetter-Bachert P, von AppenA, Hagen WJ, Beck R, Beck M, Wieland F, Briggs JAG.2015. A structure of the COPI coat and the role ofcoat proteins in membrane vesicle assembly. Science349:195–198. DOI: https://doi.org/10.1126/science.aab1121, PMID: 26160949Dodonova SO, Aderhold P, Kopp J, Ganeva I, Ro¨hlingS, Hagen WJH, Sinning I, Wieland F, Briggs JAG. 2017.9A˚ structure of the COPI coat reveals that the Arf1GTPase occupies two contrasting molecularenvironments. eLife 6:e26691. DOI: https://doi.org/10.7554/eLife.26691, PMID: 28621666Engel BD, Schaffer M, Albert S, Asano S, Plitzko JM,Baumeister W. 2015a. In situ structural analysis ofGolgi intracisternal protein arrays. PNAS 112:11264–11269. DOI: https://doi.org/10.1073/pnas.1515337112, PMID: 26311849Arakel and Schwappach. eLife 2017;6:e33650. DOI: https://doi.org/10.7554/eLife.33650 2 of 3Insight Vesicles Looking inside the cellEngel BD, Schaffer M, Kuhn Cuellar L, Villa E, PlitzkoJM, Baumeister W. 2015b. Native architecture of theChlamydomonas chloroplast revealed by in situ cryo-electron tomography. eLife 4:e04889. DOI: https://doi.org/10.7554/eLife.04889, PMID: 25584625Faini M, Prinz S, Beck R, Schorb M, Riches JD, Bacia K,Bru¨gger B, Wieland FT, Briggs JAG. 2012. Thestructures of COPI-coated vesicles reveal alternatecoatomer conformations and interactions. Science336:1451–1454. DOI: https://doi.org/10.1126/science.1221443, PMID: 22628556Hara-Kuge S, Kuge O, Orci L, Amherdt M, RavazzolaM, Wieland FT, Rothman JE. 1994. En blocincorporation of coatomer subunits during theassembly of COP-coated vesicles. Journal of CellBiology 124:883–892. PMID: 8132710Lanoix J, Ouwendijk J, Lin CC, Stark A, Love HD,Ostermann J, Nilsson T. 1999. GTP hydrolysis by arf-1mediates sorting and concentration of Golgi residentenzymes into functional COP I vesicles. EMBO Journal18:4935–4948. DOI: https://doi.org/10.1093/emboj/18.18.4935, PMID: 10487746Pepperkok R, Whitney JA, Gomez M, Kreis TE. 2000.COPI vesicles accumulating in the presence of a GTPrestricted arf1 mutant are depleted of anterogradeand retrograde cargo. Journal of Cell Science 113 (Pt1):135–144. PMID: 10591632Presley JF, Ward TH, Pfeifer AC, Siggia ED, Phair RD,Lippincott-Schwartz J. 2002. Dissection of COPI andArf1 dynamics in vivo and role in Golgi membranetransport. Nature 417:187–193. DOI: https://doi.org/10.1038/417187a, PMID: 12000962Weimer C, Beck R, Eckert P, Reckmann I, Moelleken J,Bru¨gger B, Wieland F. 2008. Differential roles ofArfGAP1, ArfGAP2, and ArfGAP3 in COPI trafficking.Journal of Cell Biology 183:725–735. DOI: https://doi.org/10.1083/jcb.200806140, PMID: 19015319Yang JS, Lee SY, Gao M, Bourgoin S, Randazzo PA,Premont RT, Hsu VW. 2002. ARFGAP1 promotes theformation of COPI vesicles, suggesting function as acomponent of the coat. Journal of Cell Biology 159:69–78. DOI: https://doi.org/10.1083/jcb.200206015,PMID: 12379802Arakel and Schwappach. eLife 2017;6:e33650. DOI: https://doi.org/10.7554/eLife.33650 3 of 3Insight Vesicles Looking inside the cell

Vesicles: Looking inside the cell.

Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes.Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes

Schwappach, Blanche

Arakel, Eric C.

GRO.publications (Univ. Göttingen)

Looking inside the cell

Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes.</jats:p

Vesicles: Looking inside the cell.

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