JournalofVisualizedExperimentswww.jove.comCopyright©2011JournalofVisualizedExperimentsJanuary2011|47|e2352|Page1of5VideoArticleInvivoandinvitroStudiesofAdaptor-clathrinInteractionDanielFeliciano1,JarredJ.Bultema1,AndreaL.Ambrosio1,SantiagoM.DiPietro11DepartmentofBiochemistryandMolecularBiology,ColoradoStateUniversityCorrespondenceto:SantiagoM.DiPietroatSantiago.DiPietro@colostate.eduURL:http://www.jove.com/video/2352DOI:doi:10.3791/2352Keywords:CellBiology,Issue47,clathrin,adaptor,Sla1p,pulldown,immunoprecipitation,GFP,fluorescencemicroscopyDatePublished:1/26/2011Citation:Feliciano,D.,Bultema,J.J.,Ambrosio,A.L.,DiPietro,S.M.InvivoandinvitroStudiesofAdaptor-clathrinInteraction.J.Vis.Exp.(47),e2352,doi:10.3791/2352(2011).AbstractAmajorendocyticpathwayinitiateswiththeformationofclathrin-coatedvesicles(CCVs)thattransportcargofromthecellsurfacetoendosomes1-6.CCVsaredistinguishedbyapolyhedrallatticeofclathrinthatcoatsthevesiclemembraneandservesasamechanicalscaffold.Clathrincoatsareassembledduringvesicleformationfromindividualclathrintriskelia,thesolubleformofclathrincomposedofthreeheavyandthreelightchainsubunits7,8.Becausethetriskeliondoesnothavetheabilitytobindtothemembranedirectly,clathrin-bindingadaptorsarecriticaltolinktheformingclathrinlatticetothemembranethroughassociationwithlipidsand/ormembraneproteins9.Adaptorsalsopackagetransmembraneproteincargo,suchasreceptors,andcaninteractwitheachotherandwithothercomponentsoftheCCVformationmachinery9.Overtwentyclathrinadaptorshavebeendescribed,severalareinvolvedinclathrinmediatedendocytosisandotherslocalizetothetransGolginetworkorendosomes9.WiththeexceptionofHIP1R(yeastSla2p),allknownclathrinadaptorsbindtotheN-terminal-propellerdomainoftheclathrinheavychain9.Clathrinadaptorsaremodularproteinsconsistingoffoldeddomainsconnectedbyunstructuredflexiblelinkers.Withintheselinkerregions,shortbindingmotifsmediateinteractionswiththeclathrinN-terminaldomainorothercomponentsofthevesicleformationmachinery9.Twodistinctclathrin-bindingm...
