CHAPTER 8 (12 NA EDIO EUROPEIA)

CYTOPLASMIC MEMBRANE SYSTEMS: STRUCTURE,

FUNCTION, AND MEMBRANE TRAFFICKING
OBJECTIVES
Emphasizethedynamicnatureoftheendomembranesystemwithinthecell.
Discriminatebetweenregulatedandconstitutivesecretion.
Outlineresearchtechniquesthathaveelucidatedthestructureandfunctionoftheendomembranesystem.
Clarifythehistorybehindthediscoveryanddescriptionofendomembranesystemorganelles.
Elucidatethestructureandfunctionoftheroughandsmoothendoplasmicreticulum.
Pointoutthedifferencesbetweenthesynthesesofsecretory/integralmembraneanddomesticproteins.
Outlinetheeventsinthesynthesisandtransportofmembranesthroughthecelltothemembrane.
Elucidatetheroleandsitesofglycosylationintheprocessingofsecretory/integralmembraneproteins.
Elucidatethestructure,functionandpolarizationoftheGolgicomplex.
Describetheroleofthevarioustypesofcoatedandnoncoatedvesiclesinmembranetrafficking.
Explainthesignalsusedtotargetproteinstotheirappropriatecellularlocation.
Describethestepsinvolvedintheprocessofexocytosisanditstriggers.
Describelysosomalstructureandfunctionandthediseasescausedbylysosomemalfunction.
Distinguishbetweenphagocytosis,bulkphaseendocytosisandreceptormediatedendocytosis.
Explaintheroleofreceptors,coatedpits,andclathrin,COPIandCOPIIcoatedvesiclesinthe
internalizationofextracellularmaterials.

LECTURE OUTLINE
An Overview of the Endomembrane System and Its Dynamic Nature
I.Beforethe20thcenturystainedtissuesectionshintedatanextensivemembranenetworkincytoplasm
A.1940sEMrevealeddiversearrayofmembranousstructuresincytoplasmofmosteukaryotes
1.Membraneboundvesiclesofvaryingdiameter;containingmaterialofdifferentelectrondensity
2.Longchannelsboundedbymembranesthatradiatethroughcytoplasm;formaninterconnected
networkofcanals
3.Stacksofflattened,membraneboundsacs(cisternae)
B.Thesestudies&subsequentbiochemicalstudiesshowedthateukaryoticcellcytoplasmwassubdivided
intoavarietyofdistinctmembraneboundcompartments
1.Sawdistinctorganellesindiversecellsfromyeasttohigherplantsandanimals
2.Theorganellesmayappearasstablestructures,but,infact,theyaredynamiccompartmentsthatare
incontinualflux
3.Theseorganelleshavedistinctstructures&functionsbuttogetherformanendomembranesystem;
theindividualcomponentsfunctionaspartofcoordinatedunit

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C.Mitochondria&chloroplastsarenotpartofthisinterconnectedsystem
D.Currentevidencesuggeststhatperoxisomeshaveadualorigin
1.Thebasicelementsoftheboundarymembraneprobablyarisefromtheendoplasmicreticulum,
2.Butmostofthemembraneproteins&solubleinternalproteinsaretakenupfromthecytoplasm
II.Theseorganellesarepartofdynamic,integratednetwork;materialsareshuttledbetweenpartsofcell
A.Transportvesiclesshuttlethingsbetweenorganelles;formbybuddingfromdonorcompartment
1.Vesicleimpliesasphericalshapedcarrier;cargomayalsobetransportedinirregularortubular
shapedmembraneboundcarriers
2.Butthetermvesicleisoftenused,keepinginmindthattheyarenotalwaysspherical
B.Transportvesiclesmoveindirectedmanner,oftenpulledbymotorproteinsoperatingontracksformed
bymicrotubules&microfilamentsofthecytoskeleton
C.Whentheyreachtheirdestination,theyfusewithacceptorcompartment,whichreceivesvesicles'
solublecargo&membranewrapper
D.Exhibitrepeatedcyclesofbudding&fusionthatmoveadiversearrayofmaterialsalongnumerous
pathwaystraversingthecell
III.Severaldistinctpathwaysthroughcytoplasmhavebeenidentified;theyfallintotwogroups:abiosynthetic
(secretory)pathway&anendocyticpathway
IV.Biosynthetic(secretory)pathwaysynthesisinER(protein)orGolgi(lipid,carbohydrate);alteredaspass
throughGolgi,sentfromtheretovariouslocations(membrane,lysosome,largeplantcellvacuole,etc.
A.ManymaterialsmadeinER(proteins)&Golgi(complexpolysaccharides)fatedforsecretionfromcell
B.Twotypesofsecretoryactivityconstitutive&regulated
1.Constitutivesynthesis&secretionintoextracellularspaceoccursincontinual,unregulated
manner;mostcellsdoittoformextracellularmatrix&plasmamembraneitself
2.Regulatedsecretorymaterialsareoftenstoredinlarge,denselypacked,membraneboundsecretory
granulesincellperiphery;secretedaftercorrectstimulus
a.Endocrinecellsreleasehormones
b.Pancreaticacinarcellsreleasedigestiveenzymes
c.Nervecellsreleaseneurotransmitters
C.Proteins,lipids&complexpolysaccharidesaretransportedthroughcellalongbiosyntheticorsecretory
pathway;discussionwillcenteronseveraldistinctclassesofproteins
1.Solubleproteinsdischargedfromcell
2.Integralproteinsofvariousmembranes
3.Solubleproteinsthatresidewithinvariouscompartmentsenclosedbyendomembranes(likelysosomal
enzymes)
V.Endocyticpathwaymovesmaterialsormembranesurfaceintocellfromoutsidetocytoplasmic
compartments(endosomes,lysosomes);movementdirectionisoppositetothatofsecretorypathway
VI.Proteinstargetedtospecificdestinationsthroughsortingsignalslocatedonthem&receptorsintransport
vesiclewallsthatrecognizethem(analogoustotruckscarryingdifferentcargotovarioussites)
A.Bothtransportpathwaysrequiredefinedtrafficpatterns;ensureaccuratedeliverytocorrectsites
1.Ex.salivaryglandcellproteintrafficking;salivarymucusproteins(madeinER)specificallytargeted
tosecretorygranules;lysosomeenzymes(alsomadeinER)specificallysenttolysosome
2.Differentorganellesalsocontaindifferentintegralmembraneproteins;theymustalsobetargetedto
particularorganelle(lysosome,Golgicisterna)

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B.Targetinginvolvesintegralmembraneproteins,secretoryproteins,lysosomalproteins;theyarerouted
totheirappropriatecellulardestinationbyvirtueofspecificaddresses(sortingsignals)
1.Sortingsignalsareencodedinproteinaminoacidsequenceorinattachedoligosaccharides
C.Sortingisfacilitatedbyspecificmembranereceptorsforsortingsignalsfoundinparticularmembranesof
endomembranesystemorbycoatsthatformonoutersurfacesoftransportvesicles
1.Specificreceptorsareonsurfacesofbuddingvesicles
2.Ensuresthatproteinistransportedtotheappropriatedestination
3.Formostpart,machineryresponsiblefordrivingthiscomplexdistributionsystemconsistsofsoluble
proteinsthatarerecruitedtospecificmembranesurfaces
4.Atthosesurfaces,theyperformtheirdesignatedactivities
D.Greatadvancesinexperimentalapproacheshavebeenmadeoverlast2or3decadesin:
1.Mappingthetrafficpatternsthatexistineukaryoticcells
2.Identifyingthespecificaddresses&receptorsthatgoverntheflowoftraffic
3.Dissectingthemachinerythatensuresthatmaterialsaredeliveredtoappropriatecellularsites
A Few Approaches to the Study of Cytomembranes
I.EMmicrographsgivedetailedviewofcellcytoplasm,butlittleinsightintofunctionsofthestructures
A.Cellsperformdynamicprocesses,butEMportraysonlystaticscenes
B.Determiningfunctionsofcellorganellesrequirednewtechniques&innovativeexperiments
C.Suchinnovationsresultedin1974NobelPrizefor3cellbiologists:ChristianDeDuve(Universityof
LouvaininBelgium),AlbertClaude&GeorgePalade(bothofRockefellerUniversity)
II.Insightsgainedfromautoradiographycandetectlocationofradioactivelylabeledmaterialsincell
A.Pancreasacinarcellshaveaparticularlyextensiveendomembranesystem;idealforstudyby
autoradiography
1.Thecellsfunctionprimarilyinsynthesis&secretionofdigestiveenzymes
2.Enzymesareshippedviaductsfrompancreas,wheretheyaresynthesized,tosmallintestineto
degradeingestedfoodmatter
B.JamesJamieson&GeorgePaladeworkedwithpancreasacinarcells;followedsecretoryproteinfrom
synthesistosecretion&determinedindividualstepseventhoughallofthemoccurredsimultaneously
1.Abletoobservestepsofsinglecycleofsecretionfromstarttofinish
2.Autoradiographyallowsvisualizationofbiochemicalprocessesbyallowinginvestigatortodetermine
thelocationofradioactivelylabeledmaterialswithincell
C.Proceduresectiontissuescontainingradiolabel&locatehotdigestiveenzymeswithautoradiography
1.Incubatetissuesliceswithhot(radioactive)aminoacidsbriefly>incorporatedintodigestiveenzymes
astheyaremadeonribosomes
2.Fixtissues;tissuesectionscontainingradioactiveisotopeswerethencoveredwiththinphotographic
emulsionlayer,whichisthusexposedtoradiolabelemanatingfromradioisotopeswithintissue
3.Sitesincellwithradiolabelarehighlightedwithdevelopedsilvergrainsinoverlyingemulsion
4.Iflabel,wash&harvestimmediately,labelappearsfirstoverRER>RERwassiteofsynthesis
III.Insightsfrompulsechasetrials(Palade&Jamieson)showsecretoryproteinpathaftersynthesistotheir
siteofdischarge
A.Exposetohotaminoacidsbriefly(pulse)followedbyawashtoremoveexcessisotopefromtissue
1.Pulsereferstothebriefincubationwithradioactivityduringwhichlabeledaminoacidsare
incorporatedintoprotein

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B.Transferredtissuetomediumwithunlabeledaminoacids(chase),whichlastsforvaryingtimeperiods
1.Duringthisperiod,proteinsynthesiscontinuesusingnonradioactiveaminoacids
2.Thelongerthechase,thefartherthehot(radioactive)proteinsmadeduringthepulsewillhave
traveledfromtheirsynthesissite(theRER)withinthecell
C.Onecanseewaveofradioactivitymovingthroughcell,discernpathwaysequenceRERwassynthesis
site&seerestofpathwayfromonelocationtothenextuntiltheprocessiscomplete
1.Definedthesecretory(biosynthetic)pathway&tiedanumberofseeminglyseparatemembranous
compartmentsintoanintegratedfunctionalunit
IV.Insightsgainedfromuseofgreenfluorescentprotein(GFP) scientistscanfollowwithintheirowneyesthe
dynamicmovementsofspecificproteinsastheymovewithinsinglelivingcell;donothavetokillcells
A.GFPissmallproteinfromcertainjellyfishthatemitsagreenfluorescentlight
1.Itsgenehasbeenisolated&canbefusedtoDNAencodingproteintobestudied
2.Theresultingchimeric(composite)DNAisintroducedintocellsthatcanbeobservedinscope
3.Onceinsidecell,chimericDNAexpresseschimericproteinconsistingofGFPfusedtoendofprotein
tobestudied
4.Usually,GFPstucktoendofaproteinhaslittleornoeffectonitsmovementorfunction&protein
understudyhasnoeffectonfluorescenceofattachedGFP
B.Example:infectcellswithvesicularstomatitisvirus(VSV)straininwhichaviralgene(VSVG)isfusedto
GFPgene;virusesusefulsincetheyturncellsintofactoryforproducingviralproteins
1.Theseviralproteinsarecarriedlikeanyotherproteincargothroughthebiosyntheticpathway
2.CellbeginstomakemassiveamountsofVSVGproteininRER
3.VSVGthengoestoGolgicomplex&eventuallytotheplasmamembraneoftheinfectedcellwhere
theyareincorporatedintoviralenvelopes
4.Canseerelativelysynchronouswaveofproteinmovement(greenfluorescence)soonafterinfection
5.SynchronyisenhancedbyuseofviruswithmutantVSVGproteinthatcannotleaveERofinfected
cellsgrownatelevatedtemperature(40C)
6.Whentemperatureisloweredto32C,thefluorescentGFPVSVGproteinthathadaccumulatedinER
movessynchronouslytoGolgicomplexforvariousprocessingevents&thentomembrane
7.Mutantsofthistypethatfunctionnormallyatreduced(permissive)temperature,butnotatelevated
(restrictive)temperaturesaredescribedastemperaturesensitivemutants
V.Insightsgainedfromthebiochemicalanalysisofsubcellularfractionscellhomogenization&organelle
isolationtechniqueswerepioneeredbyAlbertClaude&ChristianDeDuve(1950s&1960s)
A.Homogenizecells;formcytoplasmicmembranefragments,theendsofwhichfusetoformspherical
vesicles(<100nmdia)
B.Vesiclesformedfromdifferentorganelles(nucleus,mitochondrion,plasmamembrane,ER,etc.)have
variedproperties,whichallowtheirseparation(cellfractionation)fromoneanother
1.Endomembranesystem(primarilyER,Golgi)vesiclesformheterogeneous,similarsizedvesicles
(microsomes);rapidly(&crudely)purified,thenseparatedfurther;oftenretainbiologicalactivity
2.Fractionatemicrosomesintosmooth&roughmembranefractionsbygradienttechniques(Ch.18)
3.Onceisolated,onecandeterminethebiochemicalcompositionofvariousfractions
C.Exampleofuses&findingsvesiclesfromdifferentpartsofGolgiwerefoundtohaveenzymesthatadd
differentsugarstotheendsofgrowingCHOchainsofglycoproteinorglycolipids
1.Purifytheseenzymesfromthemicrosomalfraction;usethemasantigenstomakeantibodies&attach
goldparticlestotheantibodies,locationsofwhichinGolgimembranescanbeseeninEM
2.RevealedroleofGolgicomplexinstepwiseassemblyofcomplexcarbohydrates

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D.Example:identificationofproteinsincellfractionstakentonewlevelusingsophisticatedproteomic
technology;isolateorganelle,extract&separateproteins&thenidentifythembymassspectrometry
1.Hundredsofproteinscanbeidentifiedsimultaneously,providingacomprehensivemolecularportrait
ofanyorganellethatcanbepreparedinarelativelypurestate
2.Forexample,asimplephagosome,containinganingestedlatexbeadhad>160differentproteins,
manyofwhichhadneverbeforebeenidentifiedorwerenotknowntobeinvolvedinphagocytosis
3.SeveralproteinswereincludedthatwerecharacteristicofER,leadingtonewappreciationoftheER's
roleinphagocytosis
VI.Insightsgainedfromuseofcellfreesystemsisolatedpartsofcellstudiedfortheircapabilities
A.Thesecellfreesystems(whichdonotcontainwholecells)provideinformationaboutcomplexprocesses
thatwereimpossibletostudyusingintactcells
B.GeorgePalade,PhilipSiekevitz,etal.(RockefellerUniversity,1960s)studiedpropertiesofrough
microsomalfraction
1.Strippedroughmicrosomalpreparationofitsattachedparticles&foundthatisolatedparticles
(ribosomes)couldsynthesizeproteinswhenprovidedwiththerequiredcytosolingredients
2.Newlysynthesizedproteinswerereleasedintotheaqueousfluidintesttube
3.Whensameexperimentswereconductedwithcompleteroughmicrosomalfraction,theproteinswere
notreleasedintoincubationmediumbutweretrappedwithinmembranousvesiclelumens
4.Somicrosomalmembranewasnotneededforproteinsynthesis,butforsequesteringnewlymade
secretoryproteinswithinERcisternalspace
C.Overthepastfewdecades,cellfreesystemshavebeenusedtoidentifytherolesofmanyoftheproteins
involvedinmembranetrafficking;examplebelowofbuddingfromliposomes
1.Cellfreeliposomes(vesicleswhosewallsconsistofanartificialbilayercreatedfrompurified
phospholipids)usedtostudyspecificrolesofproteinsinvolvedinbudding
2.Incubateliposomeswithpurifiedproteinsthatnormallycomprisecoatsofcelltransportvesicles
3.Withoutaddedcoatproteins>novesiclebudding;withit>getbudding
4.Suchreconstitutionofcellularprocessesinvitrohasbeenusefulinthis&otherstudies
4.Theycoulddeterminetheproteinsthatbindtothemembranetoinitiatevesicleformation,those
proteinsresponsibleforcargoselection&thosethatseverthevesiclefromthedonormembrane
VII.Insightsgainedfromstudyofmutantsamutantisanorganism(orculturedcell)whosechromosomes
containoneormoregenesthatencodeabnormalproteins
A.Mutantgeneproductsvaryfromthenormal;theycancauseacharacteristicdeficiencyinthecellcarrying
themutation,whichisanalyzed
1.Determiningtheprecisenatureofdeficiencygivesinformationonfunctionofthenormalprotein
B.RandySchekman,etal.,Univ.ofCa.Berkeleystudiedgeneticbasisofsecretionusingyeastcells
1.Whyheusedyeastcellsfewgenes,small,singlecelled&abletobeculturedeasily,canbegrownas
haploidsomutantsseen;haploidformajorityoflifecycle;allowseasierdeficiencydetection
2.Genemutationinhaploidyieldsobservableeffect;cantmaskpresenceofabnormalgenewith
normalone
3.YeastERsimple&directlyconnectedtooutermembraneofnuclearenvelope;vesiclesbudfrom
ER,traveltoGolgicisternaewheretheyfuse
4.Findgenesinvolvedinsecretorypathwaybyscreeningformutantcellswithabnormaldistributionof
cytoplasmicmembranes(SECgenes)
5.FoundmutationingeneforproteininvolvedinvesicleformationatERmembrane>inabsence
ofvesicleformation,cellsaccumulatedexpandedERcisternae

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6.Foundanothermutationingeneencodingaproteininvolvedinvesiclefusion>ifthisgeneis
defective,cellsamassanexcessnumberofunfusedvesicles
7.Manymutantsthatdisruptsecretorypathwayhavebeenfound,cloned&sequenced;mutantproteins
havebeenisolated;homologousproteins(withrelatedsequences)foundinmammals
VIII.Lessonslearnedfromthesetechniques
A.Dynamicactivitiesofendomembranesystemsarehighlyconserved
B.Processessimilarinallorganisms(yeast,plant,insect&humancells);donewithremarkablysimilar
proteins(despitetheirstructuraldiversity,thesecellshaveunderlyingmolecularsimilarities)
1.Someproteinsdoingsimilarthingsindifferent(oftenwidelydivergent)speciesareinterchangeable
2.Mammaliancellfreesystemscanoftenuseyeastproteinstofacilitatevesicletransport
3.Researcherscan"cure"yeastbiosyntheticpathwaymutantsbygeneticallyengineeringthemtocarry
normalmammaliangenes
The Endoplasmic Reticulum (ER): Background Information and General
Functions
I.Historyandgeneraldescriptionfirstdetectedin19thcentury
A.Vaguecytoplasmicnetworkseeninstainedcells(ergastoplasm)
1.Inpancreascells,ergastoplasmseentodisappearuponstarvation&reappearwhenanimalfed
2.Concludedergastoplasminpancreasmakesdigestivejuices
B.LaterseeninEMbyPorterwhorenameditendoplasmicreticulum
II.Endoplasmicreticulum(ER)isdividedinto2broadcategoriesrough&smooth;bothenclosespaceso
cytoplasmdividedintocytosolic&luminal(cisternal)space;contentsofthe2spacesarequitedifferent
A.Fluorescentlylabeledproteins&lipidscandiffusefromonetypeofERintotheother,indicatingthat
theirmembranesarecontinuous
1.The2typesofERsharemanyofthesameproteins&engageincertaincommonactivities(synthesis
ofcertainlipids&cholesterol)
2.Atthesametime,numerousproteinsarefoundonlyinoneortheothertypeofDNA
3.Thus,RER&SERhavedifferentstructures&functions,whichcanbetracedtothepresenceof
differentproteinsinthe2compartments
B.SmoothER(SER)typicallytubular;interconnectingpipelinesystem;curvesthroughcytoplasm;lacks
associatedribosomes;whencellsarehomogenized,itfragmentsintosmoothsurfacedvesicles
C.RoughER(RER)extensiveorganellewithribosomesattachedtoRERoncytosolicsurface;made
mostlyofcisternae(interconnectednetworkofflattenedsacs);spaceinsideappearscontinuous
1.RERiscontinuouswithnuclearenvelopeoutermembrane(ithasribosomesoncytosolicsurface)
2.Whencellishomogenized,RERfragmentsintoroughsurfacedvesicles
3.Becausetheyhavedifferentdensities,rough&smoothvesiclescanbereadilyseparatedbydensity
gradientcentrifugation&thenstudied
D.DifferentcelltypescontainvaryingamountsofeitheroneERtypeorother;dependsoncellactivities
1.Cellsthatsecretelargeamountsofproteins(pancreasorsalivaryglandcells)>lotsofRER
III.SmoothERfunctionsextensivelydevelopedinmanycells(skeletalmuscle,kidneytubules,steroid
producingendocrinecells);itsspecificproteinsvarycelltocelldependingonfunctionsofcellsSER
A.Synthesisofsteroidhormonesingonad&adrenalcortexendocrinecells
B.Detoxificationinliverofmanyorganiccompounds(barbiturates&ethanol),whosechronicusecanlead
toSERproliferationinlivercells;detoxificationcarriedoutbyoxygentransferringenzymes

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1.Theseoxygenases,likecytochromeP450s,convertthesecompoundsintomorehydrophilic
derivativessothattheycanbemoreeasily&readilyexcreted
2.Sometimestheoxygenasescreatecarcinogens;relativelyharmlessbenzo[a]pyreneformedwhenmeat
charredonagrillisconvertedintopotentcarcinogenbySERdetoxifyingenzymes
3.Suchenzymeshavelowsubstratespecificity;oxidize1000sofdifferenthydrophobiccompounds
4.CytochromeP450smetabolizemanyprescribedmedications;geneticvariationintheseenzymes
amonghumansmayexplaindifferencesbetweenpeopleindrugeffectiveness&sideeffects
C.SequesteringCa2+ionswithincisternalspace;theirreleasetriggersspecificcellactivities
1.SERcontainsahighconcentrationofCa 2+bindingproteins
2.RegulatedreleaseofCa 2+ionsfromSERtriggersspecificcellularresponses,likeskeletalmusclecell
contraction&fusionofsecretoryvesicleswiththeplasmamembrane
III.RoughERfunctionspredominantlyexportormembraneproteinsynthesis(pancreaticacinarcells,mucus
secretingcellsofdigestivetractlining;earlystudiesdoneonthesecells)
A.Organellesofproteinsecreting,glandularepitheliumcellsaredistinctlypolarizedalongcelltallaxis
(frombasaltoapicalend);reflectsflowofsecretoryproductsfromsynthesistodischarge
1.Nucleus&extensiveRERcisternaefoundnearcellbasalsurfacenearbloodsupply;RERissiteof
synthesisproteins,carbohydratechains&phospholipidsthatmovethroughcytomembranesystem
2.Golgicomplexislocatedincentralregionofcell
3.Apicalsurfacefacesductlumenthatwillcarrysecretoryproductoutoforgan
4.Cellapicalendcontainsmembraneboundsecretoryvesicleswhosecontentsarereleasedupon
arrivalofappropriatesignal
B.ItwasfoundthatRERissecretoryproteinsynthesissite(startingpointofbiosyntheticpathway)in
pancreaticacinarcells
1.Otherexamplesfoundlaterintestinalgobletcells(secretemucoproteins),endocrinecells
(polypeptidehormones),plasmacells(antibodies),livercells(bloodserumproteins)
The Endoplasmic Reticulum (ER): Synthesis of Proteins on Membrane-Bound vs.
Free Ribosomes
I.Furtherexperimentsrevealedthatpolypeptidesaresynthesizedat2distinctlocaleswithincell
A.SomeproteinsaremadeonribosomesattachedtocytosolicsurfaceofRERmembranes
1.Proteinssecretedfromcells
2.Integralmembraneproteins
3.Solubleproteinsthatresidewithincompartmentsofendomembranesystem(ER,Golgicomplex,
lysosomes,endosomes,vesicles,plantvacuoles)
B.Otherpolypeptidesmadeonfreeribosomes(notattachedtoER)&thenreleasedintocytosol
1.Proteinsdestinedtoremainincytosol(enzymesofglycolysis,cytoskeletonproteins)
2.Peripheralproteinsofinnercellmembranesurface(spectrins,ankyrins;weaklybondedtomembrane's
cytoplasmicsurface)
3.Proteinsthataretransportedtonucleus
4.Proteinstobeincorporatedintoperoxisomes,chloroplasts,mitochondria;latter2groupsmadein
cytosol&importedfullyformed(posttranslationally) acrossmembraneintoappropriateorganelle
II.Whyareproteinsmadeatdifferentcellsites&howaretheyidentified?SignalHypothesis;earnedNobelfor
Medicine(1999);GnterBlbel,DavidSabatini&BernhardDobberstein(RockefellerU.,early1970s)
A.Suggested&demonstratedthatthesiteofproteinsynthesisisdeterminedbyinformation(aminoacid
sequence)containedinNterminalportionofprotein(firstparttoemergefromribosome)

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1.SecretoryproteinshaveNterminalsignalsequencethatdirectsemergingprotein&ribosometoER
2.SignalsequencetriggersattachmentofproteinmakingribosomestoER&proteinmovementinto
cisternalspacethroughproteinlined,aqueousERchannelasitisbeingmade(cotranslationally)
B.SometransportintoERisposttranslationalproteinismadetotallyincytosol&thenimportedintoER
1.Goesthroughsamechannelsasincotranslationalpathway;similartomechanismofmitochondrial&
peroxisomaltransport
2.PathwayisusedmuchmoreheavilyinyeastthaninmammaliancellsforimportintoER;yeastcan
survivewithoutcotranslationaltransporteventhoughtheygrowmoreslowlythannormalcells
C.Signalhypothesishasbeensubstantiatedbyalargebodyofexperimentalevidence
1.Blbel'sconceptthatproteinscontaintheirown"addresscodes"hasbeenshowntoapplyinprinciple
tovirtuallyalltypesofproteintraffickingpathwaysthroughoutcell
III.Stepsinsynthesisofsecretory/lysosomal/plantvacuolarproteinonmembraneboundribosomes
A.mRNAforsecretory/lysosomal/plantvacuolarproteinbindstofreeribosome(sameasthoseusedfor
domesticproteins)frompool;theseribosomesarenotattachedtoacytoplasmicmembrane
B.Nterminalaminosemergefromribosomewithsignalsequence(615hydrophobicaminoresidues);
targetsnascentpolypeptide&ribosomeforER
1.ThesignalsequencetargetsthenascentpolypeptidetotheERmembrane(anascentpolypeptideisone
intheprocessofbeingsynthesized&thusisnotyetfullyassembled)
2.SignalsequenceleadstocompartmentalizationofpolypeptidewithinERlumen
3.SignalisusuallyfoundatornearNterminus,butoccupiesaninternalpositioninsomepolypeptides
C.Signalsequenceisrecognizedbysignalrecognitionparticle(SRP)asitexitsribosome;SRPin
mammaliancellsconsistsof6distinctpolypeptides&asmallRNAmolecule(the7SRNA)
1.SRPbindstonascentpolypeptide'ssignalsequence&ribosome(Step1),temporarilyarrestingfurther
synthesisofpolypeptide
D.BoundSRPservesastagallowingentirecomplex(SRPribosomenascentpolypeptide)tobindtoSRP
receptoronERcytosolicsurfacespecifically;thisbindingoccursthroughatleast2distinctinteractions
1.FirstdistinctinteractionisbetweenSRP&SRPreceptor
2.Theotherinteractionisbetweenribosome&translocon
E.ThetransloconisaproteinlinedchannelembeddedintheERmembranethroughwhichthenascent
polypeptideisabletomoveinitspassagefromthecytosoltotheERlumen
1.Prokaryotictranslocon3DstructurewasdeterminedbyXraycrystallography&revealedpresenceof
aporewithintransloconinshapeofanhourglass
2.Theporehadaringof6hydrophobicaminoacidssituatedatitsnarrowestdiameter
3.Intheinactive(nontranslocating)state,whichwasthestateinwhichthestructurewascrystallized,
theopeningintheporeringispluggedbyashorthelix
4.Thisplugisproposedtosealthechannel,preventingtheunwantedpassageofcalcium&otherions
betweenthecytosol&theERlumen
F.OncetheSRPribosomenascentchaincomplexbindstotheERmembrane(Step2),theSRPis
releasedfromitsERreceptor&theribosomeisattachedtotranslocon'scytosolicend&then
1.Thenascentpolypeptide'ssignalsequenceisinsertedintothetranslocon'snarrowaqueouschannel
(Step3)
2.Itisproposedthatcontactofsignalsequencewiththetransloconinteriorleadstodisplacementofthe
plug&openingofthepassageway
G.Growingpolypeptideisthentranslocatedthroughhydrophobicporering&intoERlumen(Step4)
1.Theporeringseenincrystalstructurehasadiameter(58),considerablysmallerthanthatofa
helicalpolypeptidechain,soitispresumedthatporeexpandsasnascentchaintraverseschannel

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2.Expansionispossiblebecausetheresiduesthatmakeuptheringaresituatedondifferenthelices
H.Upontranslationtermination&completedpolypeptide'spassagethroughtranslocon,themembrane
boundribosomeisreleasedfromERmembrane;helicalplugisthenreinsertedintotransloconchannel
IV.GTPisinvolvedinsecretoryproteinsynthesisseveralstepsareregulatedbyitsbindingorhydrolysis
A.Gproteins(GTPbindingproteins)playkeyregulatoryrolesinmanydifferentcellularprocesses

1.Gproteinsexistinatleast2alternateconformations:activeGTPbound&inactiveGDPboundform;
the2conformationshavedifferentabilitiestobindotherproteins
2.Thus,Gproteinsactlikemolecularswitchesturningspecificprocessesonandoff;theGTPbinding
proteintypicallyturnsprocesson&hydrolysisofboundGTPtoGDPturnsprocessoff
3.AlsoGTPbindingproteinsgenerallyrequireaccessoryproteinstocarryouttheirfunction
B.SRP&SRPreceptor(2majorinteractantsintheaboveprocess)arebothGproteins(unusual)
1.HydrolysisofGTPboundtothesetwoproteinsoccursbetweensteps2&3&triggersthereleaseof
thesignalsequencebytheSRP&itsinsertionintothetranslocon
The Endoplasmic Reticulum (ER): Processing of Newly Synthesized Proteins in
the Endoplasmic Reticulum
I.AsnascentpolypeptideentersRERcisterna, itisacteduponbyavarietyofenzymeslocatedwithineitherthe
membraneorlumenoftheRER
A.SignalpeptideonNterminusofnascentpolypeptideisremovedfrommostofthenascentproteinsbya
proteolyticenzyme,thesignalpeptidase
B.Carbohydratesareaddedtonascentproteinbyenzymeoligosaccharyltransferase
1.Bothsignalpeptidase&oligosaccharyltransferaseareintegralmembraneproteinsresidinginclose
proximitytotranslocon
2.BothenzymesactonthenascentproteinsastheyentertheERlumen
II.TheRERisamajorproteinprocessingplant
A.Tomeetitsobligations,RERlumenispackedwithmolecularchaperonesthatrecognize&bindto
unfoldedormisfoldedproteins&givethemopportunitytoattaintheircorrect(native)3Dstructure
B.TheERlumenalsocontainsanumberofproteinprocessingenzymes,likeproteindisulfideisomerase
(PDI)
1.ProteinsenterERlumenwiththeircysteineresiduesinthereduced(SH)state,buttheyleavethe
compartmentwithmanyoftheseresiduesjoinedtooneanotherasoxidizeddisulfides(SS)
2.Theformation(&rearrangement)ofdisulfidebondsiscatalyzedbyPDI
3.Disulfidebondsplayanimportantroleinmaintainingthestabilityofproteinsthatarepresentatthe
extracellularsurfaceoftheplasmamembraneorsecretedintotheextracellularspace
III.TheERisideallyconstructedforitsroleasaportofentryforthebiosyntheticpathwayofthecell
A.Itsmembraneprovidesalargesurfaceareatowhichmanyribosomescanattach(anestimated13
million/livercell)
B.ERcisternaelumenprovideslocalenvironmentthatfavorsproteinfolding&assembly
C.ERcisternaelumenalsoprovidesacompartmentinwhichsecretory,lysosomal&plantcellvacuolar
proteinscanbesegregatedfromothernewlymadeproteins
1.ThissegregationofnewlymadeproteinsinERcisternaeremovesthemfromcytosol
2.Italsoallowsthemtobemodified&dispatchedtowardtheirultimatedestination,whetheroutsidethe
cellorwithinoneofthecytoplasm'smembranousorganelles

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The Endoplasmic Reticulum (ER): Synthesis of Integral Membrane Proteins on

Membrane-Bound Ribosomes
I.Integralmembraneproteins(otherthanthoseofmitochondria&chloroplasts)arealsosynthesizedon
membraneboundribosomesofER
A.ThesemembraneproteinsaretranslocatedintoERmembraneastheyaresynthesized(cotranslationally)
usingthesamemachineryusedforsynthesisofsecretory&lysosomalproteins
1.Unlikesolublesecretory&lysosomalproteins,however,whichpassentirelythroughERmembrane
duringtranslocation,integralproteinscontain1hydrophobictransmembranesegments
2.Thesehydrophobictransmembranesegmentsareshunteddirectlyfromthetransloconchannelintothe
lipidbilayerhowcanthistakeplace?
B.Xraycrystallographicstudiesoftransloconshowedtranslocontohaveaclamshapedconformationwith
agrooveorseamalongonesideofthewallwherethechannelmightopen&close
1.Asproteinmovesthroughtranslocon,itisthoughtthatlateralgateinchannelcontinuallyopens&
closes;allowseachnascentpolypeptidesegmenttopartitionitselfaccordingtosolubilityproperties
2.Eachsegmentmaystayintheaqueouscompartmentwithintransloconchannelormoveintothe
surroundinghydrophobiclipidbilayercore
3.Thesegmentsofnascentpolypeptidethataresufficientlyhydrophobicwillspontaneouslydissolve
intolipidbilayer&ultimatelybecometransmembraneintegralmembraneproteinsegments
C.Thisideahasreceivedstrongsupportfrominvitrostudyinwhichtransloconsweregiventhechanceto
translocatecustomdesignednascentproteinscontainingtestsegmentsofvaryinghydrophobicity
1.Themorehydrophobicthetestsegment,thegreaterthelikelihoodthatitwillpassthroughthewallof
thetranslocon&becomeintegratedasatransmembranesegmentofthebilayer
II.SinglespanningmembraneproteinscanhaveanorientationwiththeirNterminusfacingeitherthecytosol
ortheERlumen(&eventuallytheextracellularspace)
A.Themostcommondeterminantofmembraneproteinalignmentisthepresenceofpositivelycharged
aminoacidresiduesflankingthecytosolicendofatransmembranesegment
B.Duringmembraneproteinsynthesis,theinnerliningoftransloconisthoughttoorientthenascent
polypeptidesothatthemorepositiveendfacesthecytosol
III.Inmultispanningproteins,sequentialtransmembranesegmentshaveoppositeorientations
A.Fortheseproteins,everyothertransmembranesegmenthastoberotated180beforeitcanexitthe
translocon
B.Studiesperformedwithpurifiedcomponentsincellfreesystemssuggestthatatranslocon,byitself,is
capableofproperlyorientingtransmembranesegments
C.ItappearsthattransloconismorethanasimplepassagewaythroughERmembrane;itisacomplex
"machine"thatcanrecognizevarioussignalsequences&performcomplexmechanicalactivities
The Endoplasmic Reticulum (ER): Membrane Biosynthesis in the ER
I.Membranesthoughttoariseonlyfrompreexistingmembranes(notdenovo[newentitiesfrompoolsof
proteins&lipids])
A.Membranesgrowasnewlymadeproteins&lipidsareinsertedintoexistingmembranesinER;each
compartmenthasuniquemembranes
1.MembranecomponentsmovefromERtovirtuallyeveryothercellcompartment
2.Asmembranemovesfromcompartmenttocompartmentincell,itsproteins&lipidsaremodifiedby
enzymesresidinginthecell'svariousorganelles

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3.Modificationsmakeeachcompartment'smembranesuniqueincomposition&givethemdistinct
identity
4.Thesemodificationsaredonebythesameenzymesthatmodifysecretoryproteinsthatarefreeinthe
ERlumen
B.Cellmembranesareasymmetric;the2phospholipidlayers(leaflets)havedifferentcompositions
1.AsymmetryisinitiallyestablishedinERaslipids&proteinsareinsertedpreferentiallyintoonelayer
ortheother
2.Asymmetryismaintainedwhilemembranepassesthroughcellbybudding&fusionfromone
compartmenttonext
3.ComponentsatERluminalsurfaceareonluminalsurfacesoftransportvesicles,Golgicisternae&
external(exoplasmic)surfaceofplasmamembrane
4.Similarly,componentsonERcytosolicsurfacemaintaintheirorientation&areultimatelyfoundat
internal(cytoplasmic)surfaceofplasmamembrane
II.Synthesisofmembranelipids
A.MostmembranelipidsareproducedentirelyinERmembranewithfollowingexceptions:
1.Sphingomyelin&glycolipids,thesynthesisofwhichstartsinER&iscompletedinGolgicomplex
2.Someuniquemitochondrial/chloroplastmembranelipids(madebyenzymesinthosemembranes)
B.PhospholipidsaremadebyintegralERmembraneenzymeswhoseactivesitesfacecytosol
1.Newlysynthesizedphospholipidsareinsertedintotheouter(cytoplasmic)leafletofERmembrane
2.Someofthelipidsmovetoinnerleafletaidedbyflippases(activelytranslocatethemacrossbilayer)
3.LipidsarecarriedfromERtoGolgicomplex&plasmamembraneaspartofbilayersmakingup
transportvesiclewalls
C.Membranesofdifferentorganelleshavemarkedlydifferentlipidcomposition(changesmadeas
membraneflowsthroughcell)whatfactorscontributetothesechanges?
1.Conversionofonetypeofphospholipidtoanothermostorganelleshaveenzymesthatmodifylipids
alreadypresentinmembrane(examplephosphatidylserinetophosphatidylcholine)
2.Asmembranesbud,somephospholipidspreferentiallyincludedinformingvesicle,othersexcluded
3.Phospholipidtransferproteinsmovespecificphospholipidsbetweenmembranecompartments
throughaqueouscytosol&maymovethemfromERtootherorganelles(mitochondria,chloroplasts)
The Endoplasmic Reticulum (ER): Glycosylation in the Rough Endoplasmic
Reticulum
I.MostproteinsmadeonRERareglycosylated&thusbecomeglycoproteins,whetherintegralproteinsof
membrane,solublelysosomalorvacuolarenzymesorpartsofECM
A.Carbohydrategroupshavekeyrolesinfunctionofmanyglycoproteins(e.g.,bindingsitesintheir
interactionswithothermacromolecules);alsoaidinproperfoldingofproteintowhichtheyareattached
1.Sugarsequencesthatcompriseglycoproteinoligosaccharidesarehighlyspecific
2.Sugarsequencesfrompurifiedglycoproteinareconsistent&predictablehowdetermined?
B.Howisoligosaccharidesugarsequenceassembled?catalyzedbyafamilyofmembraneboundenzymes
(glycosyltransferases)
1.Glycosyltransferasestransferspecificmonosaccharidefromanucleotidesugar
2.DonorisalwaysanucleotidesugarGDPmannose,GDPfucose,UDPgalactose,UDPN
acetylglucosamine;acceptoroftransferredsugarisgrowingendofcarbohydratechain
3.Sequenceofsugartransferduringoligosaccharideassemblydependsonthesequenceofactionof
glycosyltransferasesparticipatinginprocess

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4.Glycosyltransferasesequence,inturn,dependsonthelocationofspecificenzymeswithinthevarious
secretorypathwaymembranes
5.Thus,sugararrangementinoligosaccharidechainsofaglycoproteindependsonthespatial
localizationofcertainenzymesinthisassemblyline
II.CarbohydratechainsareattachedtoproteinbyNlinkages(asparagineNatom)orOlinkages(toserineor
threonineOorcollagenhydroxylysineresidue)ofbothsoluble&integralmembraneproteins
A.Theseoligosaccharidesdifferinaveragesize,sugarcomposition&pathofsynthesis&alsoshare
propertiesliketheirhighspecificity
B.Nlinkedbasal(core)chainsegmentisassembledonlipidcarriernotprotein;thentransferredasablock
tospecificasparagineresiduesofpolypeptideasitentersRERbyoligosaccharyltransferase
1.Lipidcarrierisdolicholphosphate;embeddedinmembrane(hydrophobicmoleculebuiltfrom>20
isopreneunits)&sugarsareaddedoneatatimebymembraneboundglycosyltransferases
2.Thispartofglycosylationprocessisessentiallyinvariant
3.Inmammaliancells,itstartswithtransferofNacetylglucosamine1phosphate&thentransferof
anotherNacetylglucosamine,then9mannose&3glucoseunitsinaprecisepattern
4.Thisblockof14sugarsisthentransferredbyoligosaccharyltransferasefromdolicholphosphateto
nascentpolypeptideasitisbeingtranslocatedintoERlumen
III.MutationsthatleadtototalabsenceofNglycosylationcausedeathofembryospriortoimplantation;
A.MutationsleadingtopartialglycosylationpathwaydisruptioninERalsocauseseriousinheriteddisorders
affectingnearlyeveryorgansystem
B.ThesediseasesarecalledCongenitalDiseasesofGlycosylation(CDGs)&theyareusuallyidentified
throughbloodteststhatdetectabnormalglycosylationofserumproteins
C.Example:Oneofthesediseases,CDG1bcanbemanagedthrougharemarkablysimpletreatment
1.Itresultsfromdeficiencyoftheenzymephosphomannoseisomerase(catalyzesconversionof
fructose6phosphatetomannose6phosphate)
2.Itsreactionisacrucialreactioninthepathwaythatmakesmannoseavailableforincorporationinto
oligosaccharides
3.Thediseasecanbemanagedbygivingpatientsoralsupplementsofmannose;firsttestedinboywho
wasdyingfromuncontrolledgastrointestinalbleeding(ausualcomplicationofthedisease)
4.Withinmonthsoftakingmannosesupplements,thechildwaslivinganormallife
IV.Someoligosaccharides,especiallyinlowereukaryotes,aresimplythecore,buttheytendtodiversifyin
morecomplexorganisms(evolutionaccompaniedbydiversificationoftheCHOsequencesonproteins)
A.Afterblockadded,coreismodifiedfirstinERwithenzymaticremovalof2of3terminalglucoseresidues
byglucosidases
B.Thissetsthestageforanimportanteventinanewlymadeglycoprotein'slife
1.Duringthisstage,theglycoproteinisscreenedbyasystemofqualitycontrolthatdetermineswhether
ornotitisfittomovetothenextcompartmentofthebiosyntheticpathway
2.Thescreeningprocessbeginswitheachglycoprotein,whichatthisstagecontainsasingleremaining
glucose,bindingtoanERchaperone(calnexinorcalreticulin)
3.RemovalofremainingglucosebyglucosidaseIIleadstoreleaseofglycoproteinfromchaperone
C.Iffoldingisincompleteorifproteinismisfolded,itisrecognized&boundbymonitoringenzyme(GT)
1.IfGTbindstotheglycoprotein,itaddsasingleglucosebacktooneofthemannoseresiduesatthe
exposedendoftherecentlytrimmedoligosaccharide
2.GTrecognizesincompletelyfoldedormisfoldedproteinsbecausetheydisplayexposedhydrophobic
residuesthatareabsentfromproperlyfoldedproteins

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3.Oncetheglucoseresidueisadded,thetaggedglycoproteinisrecognizedbythesamechaperones
givingitanotherchancetofoldproperly
4.Aftersometimewithchaperone,theaddedglucoseisremoved&GTcheckstheproteinagaintoseeif
ithasachieveditsproper3Dstructure(isitpartiallyunfoldedormisfolded?)
5.If3Dstructureisright,proteincontinuesonitsway;ifnot,glucoseisadded&processrepeatsuntil
eventually,theglycoproteinhasfoldedcorrectlyoritremainsmisfolded&isdestroyed
The Endoplasmic Reticulum (ER): Mechanisms That Ensure Destruction of
Misfolded Proteins
I.MisfoldedproteinsarenotdestroyedinER,butareinsteadtransportedintocytosolbydislocation
A.Itremainsunclearwhethermisfoldedproteinsaredislocatedbackintocytosolthroughtransloconsthat
broughtthemintoERorbywayofaseparatedislocationchannelofuncertainidentity
B.Onceincytosol,misfoldedproteinsaredestroyedinproteasomes,whichareproteindegradingmachines;
thisprocessensuresthataberrantproteinsarenottransportedtootherpartsofcell
1.Butthiscanhavenegativeconsequences;inseverecasesofcysticfibrosis,theplasmamembraneof
epithelialcellsislackingtheabnormalproteinencodedbythecysticfibrosisgene
2.Inthesecases,themutantproteinisdestroyedbythequalitycontrolprocess&thusfailstoreach
thecellsurface
II.Sometimes,misfoldedproteinscanbegeneratedinERataratefasterthantheycanbeexportedtothe
cytoplasm
A.Theaccumulationofmisfoldedproteins,whichisapotentiallylethalsituation,triggersa
comprehensive"planofaction"withinthecellsknownastheunfoldedproteinresponse(UPR)
B.TheERcontainssensorsthatmonitortheconcentrationofunfoldedormisfoldedproteinsinERlumen
C.Oneproposalsuggeststhatthesensorsarenormallykeptinaninactivestatebymolecularchaperones,
particularlyBiP
1.Ifcircumstancesleadtoanaccumulationofmisfoldedproteins,theBiPmoleculesintheERlumen
become"tiedup"asaresultoftheirinteractionwiththemisfoldedproteins
2.Thisrendersthem(theBiPmolecules)incapableofinhibitingthesensors;activationofthesensors
leadstoamultitudeofsignalsthataretransmittedintoboththenucleus&cytosol
3.Thisresultsintheexpressionofhundredsofdifferentgeneswhoseencodedproteinshavethe
potentialtoalleviatestressfulconditionswithintheER
D.Amongthegenesexpressedaregenesthatencode:
1.ERbasedmolecularchaperonesthatcanhelpproteinsreachthenativestate
2.ProteinsinvolvedinthetransportoftheproteinsoutoftheER
3.Proteinsinvolvedintheselectivedestructionofabnormalproteinsasdescribedabove
E.TheUPRismorethancellsurvivalmechanism;itincludestheactivationofacelldeathpathway
1.ItispresumedthattheUPRgivesthecellanopportunitytorelieveitselfofthestressfulconditions
2.Ifthesecorrectivemeasuresareunsuccessful,thecelldeathpathwayistriggered&cellisdestroyed
From the ER to the Golgi Complex: The First Step in Vesicular Transport
I.TheexitsitesofRERcisternaearetypicallysmoothsurfaced(devoidofribosomes)&serveasplaceswhere
thefirsttransportvesiclesinbiosyntheticpathwayareformed

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II.TripfromERtoGolgihasbeenvisualizedinlivingcellsbytaggingsecretoryproteinswithgreenfluorescent
protein(GFP)
A.AfterbuddingfromERmembrane,transportvesiclesareseentofusetoeachothertoformlargervesicles
&interconnectedtubulesinregionbetweenER&Golgicomplex
B.ThisregioniscalledERGIC(endoplasmicreticulumGolgiintermediatecompartment)&thevesicular
tubularclustersthatformtherearecalledVTCs
C.Onceformed,VTCsmovefartherawayfromtheERtowardGolgicomplex;otherstudiesindicatethat
thismovementoccursalongtrackscomposedofmicrotubules
The Golgi Complex
I.DiscoveredbyCamilloGolgi(Italianbiologist,1898)inventorofnewtypesofstainingproceduresthathe
hopedmightrevealtheorganizationofnervecellswithinthecentralnervoussystem
A.Onestainusedsolutionofsilvernitrateappliedtotissuethathadbeensoakedinosmium&bichromate
1.Appliedstainforseveraldaystocerebellumnervecells&sawdarklystainingreticularnetworknear
thecellnucleus;hegottheNobelPrizeinpartforthisdiscoveryin1906
2.Laterseeninothercelltypes&namedGolgicomplex;somebelieveditexistedinlivingcells,others
thoughtitwasanartifact(artificialstructureformedduringpreparationformicroscopy)
3.Fordecades,therealityofitsexistencewasthecenterofacontroversy
B.Existenceconfirmedbeyondareasonabledoubtwhenitwasclearlyidentifiedinunfixed,freezefractured
cells;itwasnoartifact
II.Characteristicmorphologyflattened,disklikemembranouscisternaewithdilatedrims&associated
vesicles&tubules(smoothmembranessofoundwithsmoothmicrosomes)
A.Cisternae(typically0.51.0mdia)arrangedinorderlystacklikepancakes;curvedresemblinga
shallowbowl;individualGolgistacksofteninterconnectedtoformribbonlikecomplex
1.Inplants,asingleGolgistackissometimescalleddictyosome
B.Usually<8cisternaearepresentperstack,butmayhaveafewtoseveral1000distinctstacks/cell;
dependsoncelltype
1.MammaliancellGolgistacksareinterconnectedbymembranoustubulestoformasingle,large
ribbonlikecomplexsituatedadjacenttothecell'snucleus
2.Vesiclesseemtobudfromaperipheraltubulardomainofeachcisterna;manyvesiclesseemtohavea
distinctproteincoat

III.Golgicisternaepolarizedcisface(entryfaceclosesttoER);transface(exitfaceatoppositeendofstack;
closertoplasmamembrane)
A.Golgicomplexisdividedintoseveralfunctionally distinctcompartmentsarrangedalongacistransaxis;
newmaterialsentercisface&passtotransfacewheretheyexitGolgicomplex
1.cismostfacecomposedofinterconnectednetworkoftubules(cisGolginetwork;CGN);CGN&
seemstobemostlyasortingstation(shipssomeproteinsonfurtherintoGolgi,somebacktoER)
2.BulkofGolgicomplexconsistsofaseriesoflarge,flattenedcisternaedividedinto3regions:thecis
cisternae,medialcisternae,transcisternae
3.Transmostfacehasdistinctnetworkoftubules&vesicles(transGolginetwork;TGN);alsosorting
station;proteinsplacedintodifferentvesicletypes(eithertomembraneorelsewhereinthecell)
B.MembranouselementsofGolgicomplexmaybesupportedmechanicallybyaperipheralmembrane
skeletonorscaffoldcomposedofavarietyofproteins,including:

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1.Membersofspectrin,ankyrin,&actinfamilies(theseproteinsarealsopresentaspartoftheplasma
membraneskeleton)
2.TheGolgiscaffoldmaybelinkedwithmotorproteinsthatdirectthemovementofvesicles&tubules
entering&exitingtheGolgicomplex
3.AseparategroupoffibrousproteinsformaGolgi"matrix"thatplaysakeyroleinthereconstruction
oftheGolgicomplexfollowingmitosis
C.Golgicomplexcompositionisnotuniformfromcistotransend;polarized;differencesincompositionof
membranecompartments(polarization)reflectsprimaryprocessingplantrole
1.Newlysynthesizedmembraneproteins(alsosecretory&lysosomalproteins)leavetheER&enterthe
Golgicomplexatitscisface&thenpassacrossthestacktothetransface
D.Astheymovealongthestack,proteinsoriginallysynthesizedinRERaresequentiallymodifiedin
specificways;forexample:
1.Partoftheprotein'slengthmaybetrimmedbyproteolyticenzymes
2.Aminoacidsmaybemodified(hydroxylationoflysine&prolineresiduesofacollagenmolecule)
3.Theprotein'scarbohydratesaremodifiedbyaseriesofstepwiseenzymaticreactions
IV.GlycosylationinGolgicomplexsynthesissiteofmostofcellscomplexpolysaccharides(animalECM
GAGs;plantcellwallpectins&hemicellulose);keyroleinglycoprotein/glycolipidCHOassembly
A.InER,glucoseresidueshadjustbeenremoved(seeabove)fromtheendsofcoreoligosaccharideofN
linkedCHOchains
1.Asnewlysynthesizedsoluble&membraneglycoproteinspassthoughcis&medialGolgicisternae,
mostofthemannoseresiduesarealsoremovedfromthecoreoligosaccharides
2.Othersugarsareaddedsequentiallybyvariousglycosyltransferasestoproduceavarietyofdifferent
oligosaccharides
B.InGolgi,asinRER,sequencesinwhichsugarsareinsertedintooligosaccharidesisdeterminedbyspatial
arrangementofspecificglycosyltransferasesthatcontactnewproteinsastheypassthrough
1.Sialyltransferase(putssialicacidatchainterminalpositioninanimalcells)isfoundintransendof
Golgistack;expectedifnewglycoproteinswerecontinuallymovingtowardthispartoforganelle
2.InER,asinglecoreoligosaccharideisassembled;inGolgicomplex,glycosylationstepscanbequite
varied,producingcarbohydratedomainsofremarkablesequencediversity
3.ProteinsinRERlacksugarsthatarenormallyaddedinmedial&transGolgicisternae
C.UnlikeNlinkedoligosaccharides,whosesynthesisstartsinER,thoseattachedtoproteinsbyOlinkages
areassembledwhollywithinGolgicomplex
V.VesiculartransportwithinGolgi;howdomaterialsmovethroughGolgi?>2contrastingtheories
A.Cisternalmaturationmodel(uptomid1980s)itwasacceptedthatcisternaeweretransientstructures;
formatcisfacebyER/ERGICvesiclefusion,traveltotransface&alteredalongtheway
1.Cisternaemature&changeincompositionastheymovethroughGolgicomplex;eachcisterna
maturesintonextcisternaalongstack(originofname)
2.Eachcisternawasthoughttophysicallymovefromthecistothetransendofthestack,changingin
compositionasitprogressed
B.Newmodelfavored(mid1980suntillate1990s)cisternaeofGolgistackremaininplaceasstable
compartmentsheldtogetherbyproteinscaffold;knownastheVesicularTransportModel
1.Cargo(secretory,lysosomal,membraneproteins)isshuttledthroughGolgistackfromCGNtoTGN
invesiclesthatbudfromonecompartment&fusewithneighboringonefartheralongstack
VI.AcceptanceofVesicularTransportModelbasedlargelyonthefollowingobservations:

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A.EachofthevariousGolgicisternaeofstackhasdistinctresidentenzymepopulation; howcouldvarious
cisternaehavesuchdifferentpropertiesifeachgaverisetonextinlineasstatedbyothermodel?
B.LargenumbersofvesiclesareseeninelectronmicrographstobudfromrimsofGolgicisternaeJames
Rothman,etal.(Stanford,1983)
1.UsingcellfreepreparationsofGolgimembranes,theyshowedthattransportvesiclescouldbudfrom
oneGolgicisterna&fusewithanotherGolgicisternainvitro
2.Formedbasisforhypothesissuggestingthatinsidecell,cargobearingvesiclesbuddedfromcis
cisternae&fusedwithcisternaederivedfromamoretranspositioninstack
VII.Bothmodelsstillhaveproponents,butconsensushasshiftedinpastfewyearsbacktocisternalmaturation
model;severalmajorreasonssummarizedbelow:
A.Cisternalmaturation(CM)modelenvisionsahighlydynamicGolgicomplexinwhichmajorelementsof
organelle,thecisternae,arecontinuallybeingformedatthecisface&dispersedatthetransface
1.Accordingtothisview,theveryexistenceoftheGolgicomplexitselfdependsonthecontinualinflux
oftransportcarriersfromtheER&ERGIC
2.AsCMmodelsays,whentransportcarrierformationfromERisblockedeitherbycelltreatmentwith
specificdrugsoruseoftemperaturesensitivemutants,Golgicomplexsimplydisappears
3.Whenthedrugsareremovedorthemutantcellsarereturnedtothepermissivetemperature,theGolgi
complexrapidlyreassemblesasERtoGolgitransportisrenewed
B.CertainmaterialsthatareproducedinER&thentravelthroughGolgicomplexcanbeshowntostay
withinGolgicisternae&neverappearwithinGolgiassociatedtransportvesicles
1.Example:fibroblaststudieslargecomplexesofprocollagenmolecules(extracellularcollagen
precursors)movefromciscisternaetotranscisternaewithouteverleavingthecisternallumen
C.Untilmid1990s,itwasassumedthattransportvesiclesalwaysmovedinforward(anterograde)
direction,fromcisorigintotransdestination,butnewevidencesaysthat
1.Somemoveinbackward(retrograde)directionfromtransdonortocisacceptormembrane
VIII.Revisedcisternalmaturationmodelacknowledgesarolefortransportvesicles,whichhavebeenclearly
showntobudfromGolgimembranes
A.Inthismodel,transportvesiclesdonotshuttlecargoinanterogradedirection,butinsteadcarryresident
Golgienzymesinretrogradedirection
1.ThismodelofintraGolgitransportisstronglysupportedbyelectronmicrographsshowingultrathin
sectionsofculturedmammaliancellsthatwerecutfromafrozenblock
2.Frozensectionsweretreatedwithantibodiesthatwerelinkedtogoldparticlespriortoexaminationin
EM;theantibodiesweremadeagainstacargoprotein(theviralproteinVSVGprotein)
3.VSVGmoleculeswerepresentwithincisternae,butabsentfromnearbyvesicles,suggestingthatcargo
iscarriedinanterogradedirectionwithinmaturingcisternaebutnotinsmalltransportvesicles
B.Inanotherexperiment,treatedgoldlabeledantibodiesthatbindtoaGolgiresidentprotein(theenzyme
mannosidaseII)>itwasfoundinboththecisternae&associatedvesicles
1.ThisstronglysupportstheproposalthatthesevesiclesareutilizedtocarryGolgiresidentenzymesina
retrogradedirection
C.TherevisedcisternalmaturationmodelexplainshowdifferentGolgicisternaeinastackcanhavea
uniqueidentity
1.TheenzymemannosidaseIIremovesmannoseresiduesfromoligosaccharides&ismostlyrestricted
tomedialcisternae
2.Itcanberecycledbackwardintransportvesiclesaseachcisternamovestowardtransendofstack
D.SomeprominentresearchersstillarguethatcargocanbecarriedbytransportvesiclesbetweenGolgi
cisternaeinananterogradedirection,sothematterisnotyetsettled

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The Types of Vesicle Transport and Their Function: Background Information

I.Materialscarriedbetweenmembranecompartmentsbyvesiclesorothertypesofmembraneboundcarriers,
whichbudfromdonormembranes&fusewithacceptormembranes
A.Mostbuddingvesiclescoveredoncytosolicsurfacebyfuzzy,electrondenselayer
1.Thedarkstaininglayerconsistsofaproteincoatformedfromsolubleproteinsthatassembleonthe
donormembranecytosolicsurfaceatsiteswherebuddingtakesplace
2.Eachcoatedbudpinchesofftoformacoatedvesicle;assemblyisinitiatedbytheactivationofa
smallGproteinthatisspecificallyrecruitedtothesite
3.Vesiclesofsimilarsize&structurecanbeformedincellfreesystems
B.Proteincoatshaveatleasttwodistinctfunctions:
1.Theyactasamechanicaldevicethatcausesthemembranetocurve&formabuddingvesicle
2.Theyprovideamechanismforselectingcomponents(&thussolublecargo)tobecarriedbyvesicle
C.Componentsselectedfortransportcaninclude:
1.Cargotobetransported(secretory,lysosomal,&membraneproteins)
2.Machineryrequiredtotarget&dockthevesicletoanacceptormembrane
D.Proteincoatsareabletomaketheseselectionsbyvirtueoftheirspecificaffinityforthecytosolic"tails"
ofintegralproteinsthatresideinthedonormembrane
E.Vesiclemembranephospholipidsalsoplayanimportantroleinselection
1.Phosphategroupscanbeaddedtodifferentpositionsofthesugarringofthephospholipids
phosphatidylinositol(PI)convertingthemintophosphoinositides
2.Thephosphorylatedringsofthesephosphoinositidesresideatthesurfaceofthemembranewherethey
canberecognized&boundbyparticularproteins
3.Differentphosphoinositidesareconcentratedindifferentmembranecompartments,whichhelpsgive
eachcompartmentaunique"surfaceidentity"
4.Theinnerleafletoftheplasmamembrane,forexample,tendstocontainelevatedlevelsofPI(4,5)P 2,
whichplaysanimportantroleinrecruitmentofproteinslikeclathrintoendocytosissites
5.AlipidspecieslikePI(4,5)P2canhaveadynamicregulatoryrolebecauseitcanberapidlyformed&
destroyedbyenzymesthatarelocalizedatparticularplaces&timeswithinthecell
II.Severaldistinctclassesofcoatedvesicleshavebeenidentifieddistinguishedbytheproteinsthatmakeup
thecoat,theirappearanceinEM,&theirroleincelltrafficking;threearethebeststudied:
A.COPIIcoatedvesiclesmovematerialsforwardfromERtoERGIC(intermediatecompartment
betweenER&Golgi)&Golgicomplex;COPisacronymforcoatproteins
B.COPIcoatedvesiclesmovematerialsinretrogradedirectionfromERGIC&Golgistackbackward
towardER
1.AlsothoughttotransportmaterialsthroughGolgifromcistotransface
2.MayplayroleintraffickingfromERtoGolgi,fromTGNtocellmembrane,betweencompartmentsof
endocyticpathway
C.ClathrincoatedvesiclesmovematerialsfromTGNtoendosomes,lysosomes&plantvacuoles
1.Alsomovematerialsfromplasmamembranetocytoplasmiccompartmentsalongendocyticpathway
2.Alsoimplicatedintraffickingfromendosomes&lysosomes
COPII-Coated Vesicles: Transporting Cargo from the ER to the Golgi Complex

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I.COPIIcoatedvesiclesarethemostrecentlydiscovered&mediatethefirstlegofjourneythroughthe
biosyntheticpathwayfromERtoERGIC&CGN
A.COPIIcoatcontainsanumberofproteinsfirstfoundinmutantyeastcellsthatcouldnottransport
materialsfromERtoGolgi;homologousproteinsfoundincoatsofvesiclesbuddingfrommammaliancell
ER
B.AntibodiestoCOPIIcoatproteinsblockvesiclebuddingfromERmembranesbuthavenoeffecton
movementofcargoatotherstagesinthesecretorypathway
II.COPIIcoatedvesiclesarethoughttobeabletoselect&concentratecertaincomponentsthattheytransport
A.ERintegralmembraneproteinsareselectivelycapturedbecausetheyinteractspecificallywithCOPII
proteinsofvesiclecoat;severaltypesofmembraneproteinsareincludedinthisgroup:
1.Enzymesthatactatlaterstagesofbiosyntheticpathway,likeglycosyltransferasesofGolgicomplex
2.Membraneproteinsinvolvedindocking&fusionofthevesiclewiththetargetcompartment
3.Membraneproteinsthatbindsolublecargo(secretoryproteins),e.g.,membraneprotein,ERGIC53,
thatbindstomannoseresiduesfoundonoligosaccharidesofcertainsecretoryproteinsinER
B.Example:ERGIC53mutationshavebeenlinkedtoaninheritedbleedingdisorder;peoplewiththe
disorderfailtosecretecertaincoagulationfactorsthatpromotebloodclotting
III.Interactionbetweenmembraneproteins(likeERGIC53)&theCOPIIcoatismediatedbysignalsequences
inthecytosolictailsofthemembraneproteins
A.ERGIC53,forexample,isrecognizedby2neighboringphenylalaninesinitscytosolictail
B.Othertypesofsolublecargoarenotselectedatthisstage&arepresentatthesameconcentrationinthe
buddingvesicleasinERlumen
1.Proteinsthatbecomeenclosedinvesiclesbutarenotspecificallyselectedforinclusionaresaidto
movebybulkflow
2.SomeoftheintegralERmembraneproteinsmayalsobecometrappedinbuddingvesicles&
transportedthroughsecretorypathwaytoplasmamembranebybulkflow
IV.AmongCOPIIcoatproteinsisasmallGprotein(Sar1);itisrecruitedspecificallytoERmembrane;like
otherGproteinsSar1playsregulatoryrole,here,itstartsvesicleformation&regulatesvesiclecoatassembly
A.First,Sar1isrecruitedtotheERmembraneintheGDPboundform&isinducedtoexchangeitsGDPfor
aGTPmolecule(Step1)
B.UponbindingGTP,Sar1undergoesaconformationalchangethatcausesitsNterminalhelixtoinsert
itselfintothecytosolicleafletoftheERbilayer(Step2)
1.Thiseventhasbeenshowntobendthelipidbilayer,whichisanimportantstepintheconversionofa
flattenedmembraneintoasphericalvesicle
2.Membranebendingisprobablyaidedbyachangeinpackingofthelipidsthatmakeupthe2leaflets
ofthebilayer
C.InStep3,Sar1GTPhasrecruited2additionalpolypeptidesoftheCOPIIcoat,whichbindasa"banana
shaped"dimmer
1.Becauseofitscurvedshape,thisdimerprovidesadditionalpressureonthemembranesurfacetohelp
itfurtherbendintoacurvedbud
D.InStep4,theremainingsubunitsoftheCOPIIcoatbindtothemembrane,andthebudisseparatedfrom
theERmembraneintheformofaCOPIIcoatedvesicle
E.Beforethecoatedvesiclecanfusewithatargetmembrane,theproteincoatmustbedisassembledandits
componentsreleasedintothecytosol

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1.DisassemblyistriggeredbyhydrolysisoftheboundGTPtoproduceaSar1GDPsubunit,whichhas
decreasedaffinityforthevesiclemembrane
2.DissociationofSar1GDPfromthemembraneisfollowedbythereleaseoftheotherCOPIIsubunits
COPI-Coated Vesicles: Transporting Escaped Proteins Back to the ER
I.COPIcoatedvesicleswerefirstidentifiedinexperimentswherecellsweretreatedwithGTPanalogues
(moleculeswithstructuressimilartoGTP)thatcannotbehydrolyzed(unlikeGTP)
A.Inthepresenceoftheseanalogues,COPIcoatedvesiclesaccumulatedwithinthecell&couldbeisolated
fromhomogenizedcellsbydensitygradientcentrifugation
1.Theyaccumulateinpresenceofanaloguebecause(likeCOPIIcoat)theircoatcontainsasmallGTP
bindingprotein(ARF1),whoseboundGTPmustbehydrolyzedbeforethecoatcandisassemble
2.ARF1(adenosylationribosefactor)is1of8distinctproteinstomakeupcompleteCOPIcoat
B.COPIcoatedvesicleshavebeenmostclearlyimplicatedintheretrogradetransportofproteinsincluding
themovementof:
1.Golgiresidentenzymesinatranstocisdirection(likemannosidaseII)
2.ERresidentenzymesfromtheERGIC&theGolgicomplexbacktotheER
C.WhetherornotCOPIcoatedvesiclesareinvolvedinanterogradeand/orretrogradetransportbetween
Golgicisternaeremainsamatterofcontroversy
II.Retaining&retrievingresidentERproteinsifvesiclescontinuallybudfrommembranecompartments,how
doeseachcompartmentretainitsuniquecomposition?
A.WhatdetermineswhetheraparticularERmembraneproteinstaysinERorgoesontoGolgicomplex?
studiessuggestproteinsaremaintainedinanorganellebyacombinationof2mechanisms:
1.Retentionofresidentmoleculesthatareexcludedfromtransportvesicles,and
2.Retrievalofescapedmoleculesbacktothecompartmentinwhichtheynormallyreside
B.Mechanismofretentioninparticularmembraneisnotunderstood
1.Retainedproteinsmaybecomepartofcomplexesthataretoolargetobeincorporatedintoabudding
transportvesicleor.
2.Membranesmayhavedifferentdomainswithdissimilarchemicalcomposition&physicalproperties
(membranemicroheterogeneity)
a.ItispossiblethattransportedmembraneproteinsmustresideinaparticularERmembranedomain
thatcanbecapturedbytheCOPIIcoat,allowingretentionofotherproteins
III.RetrievalofescapedproteinsisbetterunderstoodproteinsthatnormallyresideinER(inlumen&
membrane)haveshortaminoacidsequencesatCterminusthatserveasretrievalsequences
A.EnsurestheirreturntoERiftheyarecarriedforwardaccidentallyintransportvesicletoERGICorGolgi
complex
B.Retrievalof"escaped"ERproteinsfromthesecompartmentsisaccomplishedbyspecificreceptorsthat
capturethemolecules&returnthemtotheERinCOPIcoatedvesicles
C.SolubleproteinsofERlumen(proteindisulfideisomerase&molecularchaperonesthatfacilitatefolding)
typicallypossesstheretrievalsignal"lysaspgluleu"[KDELinsingleletternomenclature]
1.SolubleERproteinswithKDELsignalarerecognized&boundbyanintegralmembraneprotein,the
KDELreceptor,whosecytosolictailbindstoCOPIcoat,ensuringtheirreturntoER
2.IfKDELsequenceisdeletedfromERprotein,theERproteinsarenotrecovered&broughtbackto
theERcompartment,butinsteadarecarriedforwardthroughtheGolgicomplex

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3.IfascientistengineersalysosomalorsecretoryproteinincelltohaveanaddedKDELCterminus,the
proteinisreturnedtoERratherthangoingtoitsproperdestination
D.ERintegralmembraneproteins(likeSRPreceptor)haveadifferentretrievalsignalatCterminus(usually
KKXX,whereKislysine&Xisanyaminoacid);itbindstoCOPIcoat&ensuresreturn
E.Eachbiosyntheticpathwaycompartmentmayhaveitsownuniqueretrievalsignals;thisexplainsthe
maintenanceofuniqueproteincomplementsineachonedespiteconstantin/outvesiclemovement
Beyond the Golgi Complex: Sorting of Lysosomal Proteins at the TGN
I.HowdoesparticularproteinsynthesizedinERgettargetedtowardparticularcellulardestination?
A.Cellmustbeabletodistinguishamongthevariousproteinsitmanufacturesexample:pancreaticcell
1.Mustsegregatenewlymadedigestiveenzymes(secretedintoduct),fromnewlymadecelladhesion
molecules(ultimatelyresideinplasmamembrane),fromlysosomalenzymesdestinedforlysosomes
2.Sothecellsortsproteinsdestinedfordifferentsitesintodifferentvesicles,determiningdestination
B.ProteinsortingoccursinthelastoftheGolgicompartments,thetransGolginetwork(TGN),which
functionsasamajorbranchpointinthemovementofmaterialsalongthesecretorypathway
1.TheTGNisthesiteofassemblyofclathrincoatedvesicles
2.ClathrincoatsmediatecargosortingatTGN&clathrincoatedvesiclescarryhydrolyticenzymes&
membraneproteinsfromtheretoendosomes,lysosomes&plantvacuoles
II.Lysosomalproteinsorting&transportmadeonmembraneboundRERribosomes,carriedtocisGolgi
cisternaewithotherproteintypes;thisisthebestunderstoodpostGolgipathway(forlysosomalenzymes)
A.OnceinGolgicisternae,solublelysosomalenzymesrecognizedbyenzymescatalyzing2stepadditionof
phosphategrouptocertainNlinkedCHOchainmannosesugars
1.UnlikeotherglycoproteinssortedattheTGN,lysosomalenzymespossessphosphorylatedmannose
residues,whichactasrecognitionsignals
2.Thismechanismofproteinsortingwasdiscoveredthroughstudiesonhumancellsthatlackedoneof
theenzymesinvolvedinphosphateaddition
B.Lysosomalenzymeswithmannose6phosphatesignalarerecognized&capturedbymannose6
phosphatereceptors(MPRs;integralmembraneproteinsthatspantheGolgimembranes)
C.LysosomalenzymesaretransportedfromTGNinclathrincoatedvesicles;coatsofthevesiclescontain:
1.Anouterhoneycomblikelatticecomposedoftheproteinclathrin,whichformsastructuralscaffold
2.Aninnershellmadeofproteinadaptorsthatcoverthevesiclemembranesurfacefacingthecytosol;in
molecularbiology,anadaptorisamoleculethatphysicallylinks2differenttypesofmaterials
D.LysosomalenzymesareescortedfromtheTGNbyafamilyofadaptorproteinscalledGGAs
1.EachGGAmoleculehasseveraldomains,eachcapableofgraspingadifferentproteininvolvedin
vesicleformation
2.TheouterendsofGGAadaptorsbindtoclathrinmolecules,holdingtheclathrinscaffoldingontothe
surfaceofthevesicle
3.Ontheirinnersurface,GGAadaptorsbindtosortingsignalsinthecytosolictailsofthemannose6
phosphatereceptors
4.TheMPRs,inturn,bindtosolublelysosomalenzymeswithinthevesiclelumen
5.AsaresultoftheseinteractionswithGGAadaptors,MPRsinTGNmembrane&lysosomalenzymes
withinTGNlumenbecomeconcentratedintoclathrincoatedvesicles
E.AswithCOPI/COPIIvesicleformation,clathrincoatedvesicleproductionstartswithrecruitmenttothe
membraneofsmallGTPbindingprotein(ARF1),whichsetsthestageforbindingofothercoatproteins
F.OncethevesiclehasbuddedfromtheTGN,theclathrincoatislost&theuncoatedvesicleproceedstoits
destination,whichmaybeanendosome,lysosomeorplantvacuole

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G.Oncethevesiclereachesitsdestinationorganelle,theMPRsdissociatefromthelysosomalenzymes&
returntotheTGNforanotherroundoflysosomalenzymetransport
Beyond the Golgi Complex: Sorting and Transport of Non-Lysosomal Proteins
I.Membraneproteinsdestinedforplasmamembrane&secretoryproteinsdestinedforexportfromthecellare
alsotransportedfromTGN,butthemechanismsarepoorlyunderstood
A.RecentmodelmembranouscarriersareproducedastheTGNfragmentsintovesicles&tubulesof
varioussizes;thisfitswithcisternalmaturationmodel
1.CisternalmaturationmodelsuggeststhatGolgicomplexcisternaemovecontinuallytowardTGN,
wheretheywouldhavetodispersetoallowcontinuedmaturationofGolgistack
2.Proteinsthataredischargedfromthecellbyaprocessofregulatedsecretion(digestiveenzymes,
hormones)arethoughttoformselectiveaggregates
3.Theseaggregateseventuallybecomecontainedinlarge,denselypackedsecretorygranules&are
apparentlytrappedassecretorygranulesbudfromtherimsofthetransGolgicisternae&TGN
4.Secretorygranulesarethenstoredinthecytoplasmuntiltheircontentsarereleasedafterstimulationof
thecellbyahormoneornerveimpulse
II.Thetargeteddeliveryofintegralproteinstotheplasmamembraneappearstobebasedlargelyonsorting
signalsinthecytoplasmicdomainsofthemembraneproteins
A.Inpolarizedcells,membraneproteinsdestinedtoresideintheapicalportionsoftheplasmamembrane
containdifferentsortingsignalsfromthosedestinedforthelateralorbasalportion
B.Plasmamembranesofnonpolarizedcells(fibroblasts,whitebloodcells)maynotrequirespecialsorting
signals
1.SuchproteinsmaysimplybecarriedfromtheTGNtothecellsurfaceinvesiclesoftheconstitutive
secretorypathway
Beyond the Golgi Complex: Targeting Vesicles to a Particular Compartment:
Background
I.Vesiclefusionrequiresspecificinteractionsbetweendifferentmembranes
A.VesiclesfromERfusewithERGICorcisGolginetwork&notwithatranscisterna
B.Selectivefusionoccurs&isonefactorthathelpsensureahighlydirectedflowthroughthemembranous
compartmentsofthecell
II.Thewayinwhichcellstargetvesiclestospecificcompartmentsisnotwellunderstoodbutvesiclesare
thoughttohavespecificproteinsintheirmembranesgoverningtheirmovements&fusionpotential
III.Summaryofthestepsbetweenvesiclebudding&vesiclefusionisneededtounderstandthenatureofthe
proteinsinvesiclemembranescontrollingvesiclemovement&fusion
Targeting Vesicles to a Particular Compartment: Summary of Steps Between
Vesicle Budding and Fusion
I.Movementofvesicletowardthespecifictargetcompartment
A.Vesiclesmustsometimesmovelargedistancesthroughcytoplasmbeforereachingitseventualtarget;
probablydirectedbymicrotubules
B.Microtubulesactlikerailroadtrackscarryingcargocontainersalongadefinedpathwaytoa
predetermineddestination

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II.Tetheringvesiclestothetargetcompartmentmicroscopestudiesindicatethatvesiclesareoftentetheredto
apresumedtargetcompartmentlikeGolgicisterna,byextended,fibrousproteins
A.Tetheringmaybeanearlystageinprocessofvesiclefusionthatrequiresspecificitybetweenvesicle&
targetcompartment
B.MuchofthisspecificitymaybeconferredbyafamilyofsmallGTPbindingproteins(Rabs);
1.With>60differentRabgenesidentifiedinhumans,theseproteinsconstitutethemostdiversegroupof
proteinsinvolvedinmembranetrafficking
2.Moreimportantly,differentRabsbecomeassociatedwithdifferentmembranecompartments
3.Thispreferentiallocalizationgiveseachcompartmentauniquesurfaceidentity,whichisrequiredto
recruittheproteinsinvolvedintargetingspecificity
3.IntheirGTPboundstate,Rabsarethoughttorecruitspecificcytosolictetheringproteinstospecific
membranesurfaces
III.Dockingvesiclestothetargetcompartmentatsomepointduringtheprocessleadingtovesiclefusion,
membranesofvesicle&targetcompartmentbecometightlyapposedtooneanother
A.Thisisresultofinteractionbetweenthecytosolicregionsofintegralproteinsofthe2membranes
1.ThekeyproteinsthatengageintheseinteractionsarecalledSNAREs&theyconstituteafamilyof
membraneproteinswhosemembersarelocalizedtospecificsubcellularcompartments
2.SNAREsvaryalotinstructure&size,butallofthemcontainasegmentintheircytosolicdomain(a
SNAREmotif)consistingof6070aminoacidsthatformacomplexwithanotherSNAREmotif
B.SNAREsaredividedfunctionallyinto2categories:vSNAREs(incorporatedintotransportvesicle
membranesduringbudding)&tSNAREs(locatedintargetcompartmentmembranes)
C.ThebeststudiedSNAREsarethosethatmediatedockingofsynapticvesicleswiththepresynaptic
membraneduringtheregulatedreleaseofneurotransmitters
1.Presynapticnervecellmembranecontains2tSNAREs:syntaxin&SNAP25,whilethesynaptic
vesiclemembranecontainsasinglevSNARE,synaptobrevin
2.Assynapticvesicle&presynapticmembranesapproachoneanother,theSNAREmotifsoft&v
SNAREmoleculesfromapposingmembranesinteractwithoneanothertoform4strandedbundles
3.Eachbundleconsistsof2helicesdonatedbySNAP25&1helixdonatedbybothsyntaxin&
synaptobrevin
4.Together,theseparallelhelicesformatightlyinterwovencomplexthatpullsthetwoapposinglipid
bilayersintoverycloseassociation
5.Theformationofsimilar4strandedhelicalbundlesoccursamongotherSNAREsatothersites
throughoutthecell,wherevermembranesaredestinedtofuse
D.Interestingly,theSNAREsofsynapticvesicle&presynapticmembranesaretargetsoftwoofthemost
potentbacterialtoxins,thoseresponsibleforbotulism&tetanus
1.Thesedeadlytoxinsactasproteases,whoseonlyknownsubstratesareSNAREs
2.CleavageoftheneuronalSNAREsblocksthereleaseofneurotransmitters,whichcausesparalysis
IV.Fusionbetweenvesicle&targetmembranes
A.Whenartificiallipidvesicles(liposomes)containingpurifiedtSNAREsaremixedwithliposomes
containingapurifiedvSNARE,thetwotypesofvesiclesfusewithoneanotherbutnotthemselves
1.Thisfindingindicatesthatinteractionsbetweenv&tSNAREsarecapableofpullingtwolipid
bilayerstogetherwithsufficientforcetocausethemtofuse
2.Evidencesuggeststhatwhileaninteractionbetweenv&tSNAREsisrequiredforfusion,itisnot
sufficientalonetobringaboutfusionwithinacell
B.Oneviewregardingtheregulatedsecretionofneurotransmittermolecules

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1.The4strandedSNAREbundleremainslockedinaninactiveconformation
2.Vesiclesatthisstageremaindockedatthemembrane&readytodischargetheircontentsalmost
instantaneouslyoncetheyreceiveanactivatingsignalintheformofariseinCa 2+concentration
3.Regardlessofhowitisregulated,oncemembranefusionoccurs,theSNAREsthatpreviously
projectedfromseparatemembranesbecomesituatedinthesamemembrane
4.Dissociationof4strandedSNAREcomplexisachievedbydoughnutshaped,cytosolicproteincalled
NSFthatattachestotheSNAREbundle&,usingenergyfromATPhydrolysis,twistsitapart
C.Howisspecificityofthisinteractiondetermined?currentconsensusisthattheabilityofaparticular
vesicle&targetmembranetofuseisdeterminedbythespecificcombinationofinteractingproteins
1.Theproteinsincludetetheringproteins,Rabs&SNAREs;thatcanbeassembledatthatsiteincell
2.Takentogether,thesemultipleinteractionsbetweenseveraltypesofproteinsprovideahighlevelof
specificity
Exocytosis: The Terminal Stage of Secretion
I.Beststudiedexamplesofvesiclefusionaretheregulatedfusionofsecretoryorsynapticvesicleswiththe
plasmamembrane
A.Inthesecases,membranefusionproducesopeningthroughwhichvesicle(granule)contentsarereleased
intoextracellularspace
B.Thisprocessofmembranefusion&contentdischargeiscalledexocytosis;itisusuallytriggeredbya
localincreaseincalciumionconcentration
1.ThearrivalofanerveimpulseatneuronterminalknobleadstoanincreaseinCa 2+influx&
subsequentneurotransmitterdischargebyexocytosis
2.Inthiscase,fusioninneuronismediatedbycalciumbindingprotein(synaptotagmin)foundin
synapticvesiclemembrane
3.Inothertypesofcells,exocytosisisusuallytriggeredbyCa 2+releasefromcytoplasmicstores
4.InjectionofCa2+solutionsintosecretorycellsleadstowholesaleexocytosisofsecretoryproduct
II.Stepsinexocytosisnotwellunderstood
A.Cell&vesiclemembranecontactmediatedbyfusionproteinswithin&onmembranesurface;proteins
thoughttocreatecloserangecontactbetweenmembranesdestinedtointeract&fuse
B.Contactbetweenthecell&vesiclemembranesmayleadtotheformationofasmall,proteinlinedfusion
porethatrapidlydilatestoformopeningfordischarge
C.Regardlessofmechanism,whenacytoplasmicvesiclefuseswiththeplasmamembrane:
1.Theluminalsurfaceofvesiclemembranebecomespartofoutersurfaceofplasmamembraneand
2.Thecytosolicsurfaceofvesiclemembranebecomespartoftheinnersurfaceofplasmamembrane

Lysosomes
I.Lysosomemorphology&contentstypicallycontainatleast50differenthydrolyticenzymesmadeinRER
&targetedforlysosomes;lysosomesareananimalcell'sdigestiveorganelles
A.Lysosomalenzymestakentogethercanhydrolyzevirtuallyeverytypeofbiologicalmacromolecule,
resultinginlowMWproductsthatcanbetransportedacrossthelysosomalmembraneintocytosol
B.AlloftheenzymeshavepHoptimumatacidpH(acidhydrolases)suitedtothelowpHofthelysosomal
compartment;lysosomeinteriorpHis~4.6
1.Thehighinternalprotonconcentrationismaintainedbyaprotonpump(transporter;anH +ATPase)
presentinthelysosome'sboundarymembrane

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2.Lysosmalmembranesalsocontainavarietyofhighlyglycosylatedintegralproteins;the
carbohydratechainsformaliningthatmayshieldmembranefromattackbyenclosedenzymes
C.Lysosomalmorphologyisneitherdistinctivenoruniform,althoughtheyhouseapredictablecollectionof
enzymes;theyaredynamicorganellescapableofrapidfusion&fission
1.Rangeinsizefromrelativelylarge(>1mdiameter)toverysmallvesicles(2550nmdiameter)
2.Canbeirregularinshape&ofvariableelectrondensity(likethoseinKupffercell,aphagocyticcellin
theliverthatengulfsagingredbloodcells)
3.Hardtoidentifyonmorphologicalbasisalone;identifyingtraitispresenceofacidphosphatase(assay
producesleadphosphateproductvisibleinEM)
II.Lysosomalfunctions
A.Materialsbroughtintocell(protozoa,macrophages,neutrophils)fromextracellularenvironmentare
enzymaticallybrokendown;resultingnutrientscrossmembraneintocytosol;beststudiedfunction
1.Manysinglecelledorganismsingestfoodparticles,whicharedisassembledinlysosome
2.Inmammals,phagocyticcells(macrophages,neutrophils)actasscavengers,ingestingdebris&
potentiallydangerousmicroorganisms;highlyphagocyticcellsmayhaveupto1000lysosomes
3.Ingestedbacteriaareusuallyinactivatedbylow pH&thendigestedenzymatically;somearenot
4.Peptidesmadebytheaboveprocessarepostedoncellsurface;theyalertimmunesystemto
presenceofforeignagent
B.Fertilizationspermheadcontainsspecialized(modified)lysosome(acrosome),whichcontains
typicallysosomalenzymes;unusualbecauselysosomalenzymesareactiveoutsidethecell
1.Asspermnearsegg,acrosomemembranefuseswithspermplasmamembrane,releasingstored
enzymesthatdigesteggoutercovering
2.Leavesholethroughwhichadvancingspermcanreacheggsurface
C.Organelleturnover(autophagy)regulateddestructionofcell'sownorganelles&theirreplacement
1.Duringprocess,organelle(e.g.,mitochondrion)issurroundedbyadoublemembranederivedfroman
ERcisterna;outermembranethenfuseswithlysosometoproduceautophagolysosome
2.Intheautophagolysosome,theenclosedorganelleisdegraded&thebreakdownproductsaremade
availabletothecell
3.Itiscalculatedthat1mitochondrionundergoesautophagyaboutevery10minorsoinamammalian
livercell
4.Ifnutrientsupplydrops,autophagyrateincreasestoprovidemissingnutrients&thusenergy;cell
cannibalizesitsownorganellestoacquireenergytomaintainlife
D.Inrecentyears,autophagyhasalsobeenshowntohelpprotectanorganismagainstintracellularthreats
rangingfromabnormalproteinaggregatestoinvadingbacteria
1.Autophagymayevenserveasapathwayleadingtotheprogrammeddeathofmalignantcells
E.Oncedigestiveprocessinautophagolysosomeiscompleted,organelleiscalledresidualbody
1.Dependingoncelltype,residualbodycontentsmaybeeliminatedfromcellbyexocytosisorretained
withincytoplasmindefinitelyaslipofuchsingranule
2.Lipofuchsingranulesriseinnumberwithageofindividual;accumulationisparticularlyevidentin
longlivedcells(neurons)wheregranulesareconsideredamajorcharacteristicofagingprocess
Plant Cell Vacuoles
I.Asingle,membranebound,fluidfilledcentralvacuoleoccupiesupto90%ofcellvolume;theyhaveawide
spectrumofessentialfunctions

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II.Functionsofplantcellvacuoles
A.Temporarystorehouseformanycellsolutes&macromolecules(ions,sugars,aminoacids,
proteins,polysaccharides)
B.Mayalsostoreahostoftoxiccompounds(cyanidecontainingglycosides&glucosinolates)
1.Somearepartofchemicalweaponarsenalreleasedwhencellisinjuredbyherbivoreorfungusor
2.Somearebyproductsofmetabolicreactions(usedtoisolatethemfromrestofcellintovacuole
sinceplantshavenoexcretorysystem,unlikeanimals)some,likedigitalis,importantclinically
C.Generateshighturgorpressurethatpushesoutwardagainstcellwall&maintainscellshape
1.Hashighosmoticpressure,sinceionspumpedintovacuolarcompartmentbyproteins(activetransport
systems)inmembrane(tonoplast)boundingit
2.Theionconcentrationattainedismuchhigherthanthatincytoplasmorextracellularfluid
3.Becauseofvacuole'shighionconcentration,H 2Oosmosesthroughtonoplast&intovacuole
4.Hydrostatic(turgor)pressureprovidesmechanicalsupportforplantsofttissues&providesforce
neededtostretchcellwallduringcellgrowth
D.Sitesofintracellulardigestion,similartoanimallysosomes;lysosomesareabsentinplants
1.Plantvacuoleshavesomeofthesameacidhydrolasesasfoundinlysosomes&lowpH
2.LowpHmaintainedbyVtypeH+ATPaseintonoplastthatpumpsprotonsintovacuolarfluid
3.Vacuolesarealsoendpointincellsbiosyntheticpathway;followsamebasicpathaslysosome
proteins(RER>throughGolgi>sortedatGolgitransface>targetedtovacuole)

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior Background Information and Overview
I.Cellsmusttakeinmaterialsthataretoolargetopenetratethemembraneregardlessofitspermability
properties&recycleproteinsthatresideinplasmamembranetocell'sinternalcompartments
A.Bothoftheserequirementsaremetbytheendocyticpathwayinwhichsegmentsoftheplasma
membraneinvaginatetoformcytoplasmicvesiclesthataretransportedtocellinterior
B.Twoseparateprocessesofuptakeofextracellularmaterialsintocytoplasmicvesicles,whichoccurby
differentmechanismsendocytosis&phagocytosis
1.Endocytosisprimarilyaprocessbywhichthecellinternalizescellsurfacereceptors&bound
extracellularligands(uptakeoffluid,dissolvedsolutes&suspendedmacromolecules)
2.Phagocytosistheuptakeofparticulatematter
II.Terminologyhaschangedinrecentyears;in1963,C.deDuveintroducedthetermendocytosistoinclude
ingestionofparticles(phagocytosis)&uptakeoffluid&solutes(pinocytosis)
A.Sinceithasbecomeclearthatphagocytosis&pinocytosisarefundamentallydifferentactivities,theterm
pinocytosisisbeingusedlessoften
B.Forexample,phagocyticvesiclesusually~10Xlargerthanendocyticones(12mvs.0.10.2m
india)
C.Endocytosisisnowemployedtodescribetheuptakeofbothfluid&dissolvedorsuspendedmolecules;
endocytosisisdistinguishedfromphagocytosis
The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior
Introduction to Endocytosis
I.Endocytosisuptakeoffluid,dissolvedsolutes,suspendedmacromolecules;dividedinto2broadcategories:
bulkphase&receptormediatedendocytosis

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A.Bulkphaseendocytosis(alsoknownaspinocytosis)nonspecificuptakeofextracellularfluidswithout
recognitionbymembrane
1.Anymolecules(largeorsmall)thathappentobepresentinenclosedfluidaretakenintocellaswell
2.Visualizedbyaddingsubstancetoculturemedium(dyeluciferyellow;enzymehorseradish
peroxidase);takenupnonspecifically
3.Occurscontinuallyincertaincelltypeswhereitmayfunctionprimarilytoconvertplasmamembrane
intocytoplasmicmembrane;keepscellfromaccumulatingtoomuchplasmamembrane
4.Thisconversionisrequiredincellsthathavebeenengagedinsecretion&havehadlargenumbersof
secretoryvesiclesfusewiththeplasmamembrane
5.Alsoremovesportionsofplasmamembrane&mayfunctionprimarilyintherecyclingofmembrane
betweenthecellsurface&interiorcompartments
B.Receptormediatedendocytosis(RME)bringsaboutuptakeofspecificextracellularmacromolecules
(ligands)thatbindtoreceptorsonexternalplasmamembranesurface
II.Rateofbothprocessescanberemarkablyrapiduptohalfmembranesurfacecanbeinternalizedinaslittle
as30min
A.Despiterapidinwardmovementofplasmamembrane,thereisnoshrinkageofcellsurface
B.Noristhereanyimmediateneedforsynthesisofnewmembranecomponents
C.Membraneissimplycycledbetweensurface&cellinteriorsothatmembraneisaddedtosurfaceasfast
asitisremoved;exocytosisrateequalsthatofendocytosis(membraneisrecycled)
The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior
Receptor-Mediated Endocytosis and the Role of Coated Pits
I.RMEprovidesmeansforselective&efficientuptakeofmacromoleculesthatmaybepresentatrelativelylow
concentrationsinextracellularfluid
A.Cellshavereceptorsfortheuptakeofmanydifferenttypesofligands(hormones,growthfactors,
enzymes,plasmaproteins)
1.SubstancesthatentercellbyRMEbindreceptorsthatcollectinspecializedareasofplasma
membrane(coatedpits)
2.Receptorsareconcentratedincoatedpitsto1020Xthatinrestofmembrane
B.Coatedpitsmembranesurfacesitesthatareindented&coveredoncytoplasmicfacebybristly,electron
denseproteinlayercontainingclathrin&adaptors
1.ClathrinisthesameproteininclathrincoatedvesiclesformedatTGN
2.Coatedpitsinvaginateintocytoplasm&thenpinchfreeofplasmamembranetoformcoatedvesicles
II.Structureofcoatwhenviewedfromitscytoplasmicsurface,bristlycoatappearstoconsistofnetworkof
polygons(hexagons&pentagons)resemblinghoneycomb;explainsformationofcoat
A.Geometricconstructionofcoatisderivedfromstructureofitsclathrinbuildingblocks
1.Eachclathrinmoleculeconsistsof3heavy&3lightchainsjoinedtogetheratthecentertoforma3
leggedassembly(triskelion)
B.Triskelionswithintheclathrinscaffoldofacoatedvesiclearefoundinanoverlappingarrangement
1.Eachlegofaclathrintriskelionextendsoutwardalongtwoedgesofapolygon
2.Theclathrinmoleculesoverlapinsuchawaythateachvertexofapolygoncontainsacenterofone
ofthecomponenttriskelions
III.LikeclathrincoatedvesiclesbuddingfromTGN,coatedvesiclesformedduringendocytosisalsocontaina
layerofadaptorssituatedbetweenclathrinlattice&vesiclesurfacefacingthecytosol

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A.ThebeststudiedadaptoroperatinginconnectionwithclathrinmediatedendocytosisisAP2
B.UnlikeGGAadaptorsusedatTGN(consistingofasinglesubunitwithseveraldomains),AP2adaptors
incorporatedintovesiclesbuddingfromcellmembranehavemultiplesubunitswithdifferentfunctions
1.ThesubunitofAP2adaptorsengagescytoplasmictailsofspecificmembranereceptorsleadingto
concentrationoftheselectedreceptors(&boundcargomolecules)intoemergingcoatedvesicle
2.Incontrast,AP2adaptoradaptinsubunitbinds&recruitsclathrinmoleculesofoverlyinglattice
3.3leggedclathrintriskelionsoverlapwithinvesiclecoatwall,&theclathrinlattice&adaptorsinteract
C.UnlikeCOPI&COPIIvesicles,whichhaverelativelysimpleconstruction,clathrincoatedvesicleshave
upwardsof2dozendifferentaccessoryproteinsformingdynamicnetworkofinteractingmolecules
1.Theseproteinshavepoorlyunderstoodrolesincargorecruitment,coatassembly,membrane
invagination,interactionwithcytoskeletalcomponents,vesiclerelease&membraneuncoating
2.Beststudiedaccessoryproteinisdynamin
IV.DynaminisalargeGTPbindingproteinthatisrequiredforthereleaseofaclathrincoatedvesiclefrom
themembraneonwhichitforms
A.Dynaminselfassemblesintoahelicalcollararoundtheneckofaninvaginatedcoatedpit,justbeforeit
pinchesofffromthemembrane
B.HydrolysisofboundGTPbythepolymerizeddynaminmoleculesisthoughttoinduceaconformational
changeinthedynaminhelixthatseverscoatedvesiclefromtheplasmamembrane
1.SomethinkthatdynaminthusactsasanenzymethatcanutilizeGTP'schemicalenergytogenerate
mechanicalforcesthismodelhasconsiderableexperimentalsupport
2.AnothermodelsuggeststhatdynaminactsmorelikeotherGproteinsbyswitchingontheactivityofa
separateeffectorproteinthatseversthevesicle
C.Withinaminuteofitsformation,thecoatedendocyticvesiclelosesitsclathrincoat&becomesasmooth
surfacedvesiclethatenterstheendocyticpathway
The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior Overview
I.Moleculestakenintoacellbyendocytosisareroutedthroughawelldefinedendocyticpathway
II.2differenttypesofreceptorsaresubjectedtoendocytosis
A.Housekeepingreceptorsresponsibleforuptakeofmaterialsthatwillbeusedbycell;beststudied
examplesaretransferrin&LDLreceptors;mediatedeliverytocellsofiron&cholesterol,respectively
1.Endocytosisofthesereceptorsleadstypicallytothedeliveryoftheboundmaterials(likeiron&
cholesterol)tothecell&returnofthereceptortothecellsurfaceforadditionalroundsofuptake
B.Signalingreceptorsresponsibleforbindingextracellularligandsthatcarrymessagesthatchangecell
activities;theseligands(hormoneslikeinsulin;growthfactorslikeEGF)donotactuallyentercell
1.Instead,theybindtothesurfacereceptor&signalaphysiologicalresponseinsidethecell
2.Theirendocytosisleadstypicallytodestruction ofreceptor(receptordownregulation),whichhas
theeffectofreducingthecell'ssensitivitytofurtherstimulationbythehormoneorgrowthfactor
3.Receptordownregulationisamechanismbywhichcellsregulatetheirabilitytorespondto
extracellularmessengers
4.Signalingreceptorsareusuallymarkedforendocytosis&subsequentdestructionbythecovalent
attachmentofa"tag"tothecytoplasmictailofthereceptorwhileitresidesatthecellsurface
5.Thetagisasmallmolecule(ubiquitin),whichisaddedenzymatically;membraneproteinsthatare
notnormallysubjectedtoendocytosisareinternalizediftheyaremadetocarryanaddedubiquitin

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III.Endocyticpathwaybeginswithadynamicnetworkoftubules&vesiclesknowncollectivelyasendosomes
A.EndosomelumenfluidisacidifiedduetoactivityofendosomemembraneH +ATPase(H+pump)
B.Endosomesaredividedinto2distinctclassesdistinguishedfromoneanotheronbasisofbuoyantdensity
(allowsthemtobeisolatedindifferentfractionsondensitygradient),pH,proteincomposition
1.Earlyendosomestypicallylocatednearperipheralregionofcell
2.Lateendosomestypicallylocatedinmoreinteriorpartofcell,closertonucleus
C.Lateendosomesmaycontainconsiderableamountsofinternalmembranethatarisesfrominward
invaginationsoftheboundarymembrane
1.Theseinternalvesiclesoftencontainplasmamembraneproteinsonthepathtodestruction
IV.Receptorstakenupbyendocytosisaretransportedinendocyticvesiclestoanearlyendosome,whichserves
asasortingstationthatdirectsdifferenttypesofreceptors&ligandsalongdifferentpathways
A.Housekeepingreceptorsdissociatefromtheirboundligandsintheacidifiedendosomalenvironment
1.Thereceptorsarethenconcentratedintothespecializedtubularcompartmentsoftheearlyendosome,
whichrepresentrecyclingcenters
2.Vesiclesbuddingfromthesetubulescarryreceptorsbacktoplasmamembraneforadditionalrounds
ofendocytosis
B.Incontrast,releasedligands(e.g.,LDL)becomeconcentratedintoasortingcompartmentbeforebeing
dispatchedtoalateendosome&ultimatelytoalysosome,wherefinalprocessingoccurs
C.Signalingreceptorspreviouslymarkedwithubiquitintagsdonotrecyclebacktomembrane,butinstead
aresentontolateendosomes&lysosomeswheretheywillultimatelybedestroyed
V.Stepsalongendocyticpathwayfromanearlyendosometoalysosomehavebeendescribedinvariousways
bydifferentresearchersworkingondifferentcells
A.Severalreportstransferofmaterialsfromearlytolateendodomesoccursbymeansofspecialized
carriervesicles,oftenreferredtoasmultivesicularbodies(MVBs)
1.Theirnamederivesfromfactthattheyaretypicallypackedwithinternalvesicles
2.Internalvesiclesariseasinwarddirectedinvaginationsfromtheboundarymembraneofcarrier
3.Internalvesiclemembranescontainreceptors&othermembraneproteinsubiquinatedatcellsurface
4.Thissuggeststhatubiquitinservesassortingsignalthatinitiallycausesproteintobeinternalized&
subsequentlycausestheproteintoendupaspartofMVBinternalvesicles
5.MVBsmovedeeperintocellwheretheyeitherfusewithormatureintolateendosomes
6.Eitherway,lateendosomestypicallycontainconsiderableamountsofinternalmembranethatis
derivedfromMVBinternalvesicles
B.Moleculesthattravelalongendocyticpathwayinalateendosome areultimatelydirectedtoalysosome,
theterminalcompartmentoftheendocyticpathway;thismovementoccursby2majorroutes
1.Maturationoflateendosomesintolysosomesinadditiontogettingmaterialfromearlyendosomes,
lateendosomesgetnewlymadelysosomalenzymesfromTGN(carriedbyreceptors)
2.Fusionoflateendosomeswithpreexistinglysosomes
C.Onceinalysosome,membranereceptors&othermacromoleculesaredestroyed,buttransported
materialslikecholesterolaretypicallyprocessedfordeliverytothecytosol
1.Oncetheyhavedeliveredtheircargotolateendosomes,receptorsthatcarriedlysosomalenzymesto
thelateendosomesarerecycledbacktotheTGNforadditionalroundsoftransport
The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior
LDLs and Cholesterol Metabolism

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I.First&beststudiedexampleofreceptormediatedendocytosisisthemechanismthatprovidesanimalcells
withexogenouscholesterol
A.Cholesterolisahydrophobicmolecule;itservesasaprecursortosteroidhormones&anessentialpartof
plasmamembraneinanimals;can'tbefreeinblood
B.Cholesterolistransportedthroughbloodaspartofhugelipoproteincomplexes(likelowdensity
lipoprotein;LDL)
1.EachLDLparticlehascentralcore(~1500cholesterolsesterifiedtolongchainfattyacids)
2.Coreissurroundedbysinglelayerofphospholipids&asinglecopyofalargeprotein,
apolipoproteinB100,whichbindsspecificallytoLDLreceptorsoncellsurfaces
II.LDLreceptorsfoundmostlyinlivercells;transportedtocell'splasmamembranewherewaitforLDL
A.LDLreceptorsareconcentratedincoatedpitseveninabsenceofligand;thus,theyareinplasma
membrane&readytotakeupbloodbornelipoproteins,iftheyshouldbecomeavailable
1.Whenitpassesmembrane,LDLbindstocoatedpit;thepitinvaginatesformingcoatedvesicle,the
clathrincoatdisassembles,&LDLreceptorsarerecycledbacktoplasmamembrane
2.LDLparticlesgotolysosomes;proteincomponentisdegraded&cholesterolisreleasedforuseby
cell(membraneassemblyorothermetabolicprocesses,likesteroidhormoneproduction)
B.Peoplewitharareinheriteddisorder(NiemannPicktypeCdisease)lackoneoftheproteinsrequiredto
transfercholesteroloutoflysosomes
1.Theresultingaccumulationofcholesterolintheseorganellesleadstonervedegeneration&deathin
earlychildhood
III.LDL&atherosclerosis(narrowingofmajorarteries)
A.LDLbloodlevelshavebeenrelatedstronglytothedevelopmentofatherosclerosis
B.Recentstudiessuggestthatatherosclerosisresultsfromachronicinflammatoryresponsethatis
initiatedbythedepositionofLDLwithintheinnerwallsofthevessels
1.LDLdepositionleadstodevelopmentofplaquesonarterywalls
2.Plaquesreducebloodflowthroughvessel&actassitesforformationofbloodclots(can
completelyblockflow)
3.Clotsthatblockcoronaryarteriesareleadingcauseofmyocardialinfarction(heartattack)
C.LDLloweringdrugs(statins;pravastatinorlovastatin)inhibitakeycholesterolsynthesisenzyme,
HMGCoAreductase;lowersbloodcholesterol&heartattackfrequency
1.Cellsmakelesscholesterolsomusttakeupmorefromblood
2.MakemoreLDLreceptorssomoreLDLtakenup&lesscholesterolisthuspresentinblood
3.Whenbloodcholesterollevelsarelow,thefrequencyofheartattackisreduced
IV.HDLs(highdensitylipoproteins),inadditiontoLDLs,transportcholesterolinblood;similarin
construction,buthavedifferentprotein(apolipoproteinA1)&playdifferentphysiologicalroleinbody
A.LDLprimarilycarriescholesterolmoleculesfromliver,wheretheyaresynthesized&packaged,
throughbloodtobodycells;HDLcarriesexcesscholesterolfrombody'scellmembranestoliver
1.ExcesscholesterolistransportedoutofbodycellplasmamembranesdirectlytocirculatingHDL
particles
2.WhenHDLgetstoliver,itisendocytosed&cholesterolisexcretedaspartofbile
3.HDLisoftenreferredtoasthe"goodcholesterol"
B.WhilehighbloodLDLlevelsleadtoheartdisease;highbloodHDLlevelsareassociatedwithlowered
risk(goodforheart;itseemstoremovecholesterolfromblood),butthingsarenotthissimple

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1.Theenzymecholesterylestertransferprotein(CETP)transferscholesterolmoleculesfromHDLto
otherlipoproteinparticles,anactivitythattendstolowerHDLcholesterollevels
2.CETPhasbeenafocusofresearchsinceapopulationofJapanesefamilieswasfoundwhose
membersroutinelylivefor>100years&carrymutationsintheCETPgene
3.AnumberofsmallMWCETPinhibitorshavebeentestedinclinicaltrials&foundtogreatlyincrease
HDLlevelsintheblood
4.Whetherornotthisresponsecorrelateswithareducedlevelofcoronaryheartdiseaseiscurrently
underinvestigation

The Endocytic Pathway: Moving Membrane and Materials into the Cell Interior Phagocytosis
I.Phagocytosis(celleating)uptakeofrelativelylargeparticulatematter(>0.5mindia);extensiveinafew
celltypesspecializedforuptakeofparticulatematterfromenvironment&deliverytolysosomes
A.Singlecelledheterotrophs(amoebae,ciliates)maketheirlivelihoodthisway;trapfoodparticles&
smallerorganisms&enclosethemwithinfoldsofplasmamembrane,engulfingfoodparticles
1.Foldsfusetoformvacuole(phagosome)thatpinchesoffinwardlyfromplasmamembrane
2.Phagosomethenfuseswithlysosomeformingphagolysosome,withinwhichmaterialisdigested
3.Processissomewhatsimilartodigestionofcytoplasmicorganellebyautophagy
B.Inmostanimals,phagocytosisbycertaincellsisprotectivemechanismratherthanmodeoffeeding
1.Mammalspossessavarietyofprofessionalphagocytes(macrophages,neutrophils)wanderthrough
blood&tissuesphagocytizinginvaders,damaged&dyingcells,agingRBCs,debris
2.Thesematerialsarerecognized&boundbyhighlyselectivesurfacereceptorsonsurfaceof
phagocytepriortouptake;startedbycontactofcellwithrighttarget
3.Mammalianphagocytosisismarkedlyenhancedbyanumberofbloodborneproteins(opsonins)that
coattheparticletobeingested
4.Onceinsidethephagocyte,microorganismsarekilledbylysosomalenzymesoroxygenfree
radicalsgeneratedinphagosomelumen
II.Aproteomicstudyofmacrophagephagosomesrevealedthepresenceofasurprisinglylargenumberof
proteinsintheseseeminglysimplemembraneboundvacuoles
A.AmongtheproteinsfoundinphagosomemembranewereanumberofspeciescharacteristicoftheER,
includingthechaperonecalnexin
B.Itwasfoundthatmostofthephagosomemembranecontentofamacrophageisactuallyderivedfrom
ERratherthantheplasmamembrane
1.ItappearsthatinteractionofcellsurfacewithaparticletobeengulfedleadstorecruitmentofER
intotheregionjustbeneaththeplasmamembrane
2.Asparticleisengulfed,underlyingERfuseswithplasmamembrane,producingaphagosome
membranecomposedlargelyofER
3.Thisappearstobeonewaythatphagocyticcellsareabletoaddalargeamountofrequired
membranetotheircellsurfaceinashortamountoftime
III.Phagocytosisisdrivenbyactincontainingmicrofilamentcontractileactivitiesunderlyingcellmembrane

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IV.Notallbacteriaingestedbyphagocyticcellsaredestroyed;infact,somespecieshijackphagocytic
machinerytopromotetheirownsurvivalinbody
A.Mycobacteriumtuberculosistakenintomacrophagecytoplasmbyphagocytosis;phagosomedoesn't
fusetolysosome;bacteriuminhibitsfusionthatwouldkillit&multiplieswithincell
B.Coxiellaburnetii(causesQfever)itsphagosomefuseswithlysosome,butneitheracidpHnorthe
lysosomalenzymescandestroyit
C.Listeriamonocytogenes(causesmeningitis)producesproteinsthatdestroylysosomalmembrane
integritysobacteriumcanescapeintocellcytosol

Posttranslational Uptake of Proteins: Peroxisomes

I.Proteintraffickingincellisgovernedby:
A.Sortingsignalssecretedproteinsignalpeptideormannosephosphategroupsoflysosomalenzymes
B.Receptorsthatrecognizethesesignals&delivertheproteincontainingthemtopropercompartment
II.Fourofcell'smajororganelles(nucleus,mitochondria,chloroplasts,peroxisomes)importproteins
throughoneormoreouterboundarymembranes
A.Proteinsimportedbytheseorganellescontainaminoacidsequencesthatserveasaddressesrecognized
byreceptorsatorganelle'soutermembrane,aswithRER
B.UnlikeRER,whichusuallyimportsproteinscotranslationally,proteinsoftheseorganellesare
importedposttranslationally(aftertheircompletesynthesisonfreeribosomesinthecytosol)
III.Uptakeofproteinsintoperoxisomes
A.Peroxisomesareverysimpleorganelleswithonly2subcompartmentsinwhichanimportedprotein
canbeplaced:boundarymembrane&internalmatrix
B.Proteinsdestinedforperoxisomepossessperoxisomaltargetingsignal:eitheraPTSfora
peroxisomalmatrixproteinoranmPTSforaperoxisomalmembraneprotein
1.SeveraldifferentPTSs,mPTSs&PTSreceptorshavebeenidentified
2.PTSreceptorsbindtoperoxisomedestinedproteinsincytosol&shuttlethemtoperoxisome
membranepriortoimport
3.Thereceptorapparentlyaccompaniesperoxisomalproteinthroughmembraneintomatrix&then
recyclesbacktothecytosoltoescortanotherprotein
C.Unlikemitochondria&chloroplasts,whoseimportedproteinsmustassumeanunfoldedstate,
peroxisomescansomehowimportperoxisomalmatrixproteinsintheirnative,foldedconformation
1.Thisiseventrueofperoxisomalproteinsthatconsistofseveralsubunits
2.Mechanismresponsibleremainsamatterofspeculation
Posttranslational Uptake of Proteins: Mitochondria

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I.Mitochondriahave4subcompartmentsintowhichproteinscanbedelivered:
A.Outermitochondrialmembrane(OMM)
B.Innermitochondrialmembrane(IMM)
C.Intermembranespace
D.Matrix
II.Mitochondriasynthesizeafewoftheirownintegralmembranepolypeptides(13inmammals),butthevast
majorityoftheorganelle'sproteins(roughly99%)areencodedbynucleargenome
A.Theseproteinsaresynthesizedinthecytosol&importedposttranslationally
B.Proteinsofmitochondrialmatrix&IMMmakeupthevastmajorityofproteinstargetedtomitochondria
sodiscussionisrestrictedtothem
III.Aswithperoxisomalproteins&thoseofothercompartments,mitochondrialproteinscontainsignal
sequencesthattargetthemtotheirhomebase
A.Mitochondrialmatrixproteinshavetargetingsequence (presequence)foundatmolecule'sNterminus;it
includesanumberofpositivelychargedresiduesthatlieononefaceofanextendedhelix
1.TheNterminaltargetingsequenceisultimatelyremovedbyamitochondrialprocessingprotease
followingimportintomatrix
B.Incontrast,mostproteinsdestinedforIMMcontainseveralinternalsequencesthatremainaspartofthe
molecule
IV.Beforeaproteincanenteramitochondrion,severaleventsarethoughttotakeplace:
A.Proteinmustbepresentedtoamitochondrioninrelativelyextendedorunfoldedstate
B.Severaldifferentmolecularchaperonesareimplicatedinpreparingpolypeptidesformitochondrialuptake,
includingonesthatspecificallydirectmitochondrialproteinstocytosolicOMMsurface
1.Othersunfoldpolypeptides&preventtheiraggregation
V.OMMcontainsproteinimportcomplex(TOMcomplex),whichincludesareceptorthatrecognizes&
bindsmitochondrialproteins&aproteinlinedchannel
A.UnfoldedpolypeptidesaretranslocatedthroughOMMviatheproteinlinedchannel
1.UnlikeERtransloconorperoxisome,poreformingproteinofTOMcomplexisabarrelprotein,like
otherOMMintegralproteins,reflectingitsevolutionfromancestralbacteriumoutermembrane
2.Functionalconsequencesbarrelproteincannotopenlaterallytoallowintegralproteinstoinsertinto
OMM;OMMproteinsmustpassintointermembranespacebeforeenteringOMMbilayer
B.ProteinsdestinedfortheIMMormatrixmustpassthroughintermembranespace&engageasecond
proteinimportcomplexlocatedintheIMM(TIMcomplex);IMMcontains2majorTIMcomplexes
1.TIM22bindsintegralproteinsofIMM&insertsthemintolipidbilayer
2.TIM23bindsmatrixproteins&translocatesthemcompletelythroughIMMintoaqueousmatrix
compartment
C.MovementintomatrixpoweredbyelectricpotentialacrossIMMactingon"+"chargedtargetingsignal
1.IfpotentialisdissipatedbyadditionofdruglikeDNP,translocationceases&polypeptideremains
trappedwithinmembrane
VI.Aspolypeptideentersaqueousmatrix,itinteractswithmitochondrialchaperones(e.g.,mtHsp70)thatmediate
entry;2mechanismsexplainthegeneralchaperoneroleinproteinmovementacrossmembranes:
A.Accordingtooneview,chaperonesactasforcegeneratingmotorsthatuseenergyfromATPhydrolysisto
"pull"theunfoldedpolypeptidethroughthetranslocationpore

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B.Alternateviewchaperonesaidinpolypeptidediffusionacrossmembrane;thisisarandomprocessin
whichamoleculecanmoveinanyavailabledirection
1.Proteinprotrudesintomatrixthroughtranslocationpore,achaperoneresidingontheinnersurfaceofthe
membranegrabsit&keepsitfromdiffusingbackoutthroughtheporeandintothecytosol
2.Thechaperone,however,didnotblocktheprotein'sdiffusionfurtherintothematrix
3.Aspolypeptidediffusesfurtherintomatrix,itbindsrepeatedlytothechaperone&ateachstage
preventedfromdiffusingbackwardoutofmatrix;mechanismcalledbiaseddiffusion
4.ThechaperoneissaidtobeactingasBrownianratchet(thetermBrownianimpliesrandomdiffusion;
aratchetisatoolthatallowsmovementinonlyonedirection)
C.Recentevidencesuggeststhatbothoftheabovemechanismsofchaperoneactionareprobablyused&act
cooperatively
Posttranslational Uptake of Proteins: Chloroplasts
I.Chloroplastshave6subcompartmentsintowhichproteinscanbedelivered:
A.Inner(1)&outer(2)envelopemembrane&interveningintermembranespace(3)
B.Stroma(4),thylakoidmembrane(5),thylakoidlumen(6)
II.Chloroplast&mitochondrialimportmechanismsexhibitmanysimilarities,althoughtheirtranslocation
machineryhaveevolvedindependently;asinmitochondria:
A.Vastmajorityofchloroplastproteinsareimportedfromcytosol
B.Theouter&innerenvelopemembranescontaindistincttranslocationcomplexes(Toc&Tic
complexes,respectively)thatworktogetherduringimport
C.Chaperonesaidintheunfoldingofthepolypeptidesinthecytosol&foldingoftheproteinsinthe
chloroplast,and
D.ProteinsdestinedforthechloroplastaresynthesizedwitharemovableNterminalsequence(termed
thetransitpeptide)
III.Transitpeptidedoesmorethansimplytargetapolypeptidetoachloroplast;itprovidesanaddressthat
localizesthepolypeptidetooneofseveralpossiblesubcompartmentswithintheorganelle
A.Allproteinstranslocatedthroughthechloroplastenvelopecontainastromatargetingdomainaspart
oftheirtransitpeptide;thisguaranteesthatthepolypeptidewillenterthestroma
1.Onceinthestroma,thestromatargetingdomainisremovedbyaprocessingpeptidaselocatedin
thatcompartment
B.Thosepolypeptidesthatbelonginathylakoidmembraneorathylakoidlumenbearanadditional
segmentintheirtransitpeptide(thylakoidtransferdomain),thatdictatesentryintothethylakoids
C.Severaldistinctpathwayshavebeenidentifiedbywhichproteinsareeitherinsertedintothethylakoid
membraneortranslocatedintothethylakoidlumen
1.Thesepathwayshavestrikingsimilaritiestotransportsystemsinbacterialcells,thepresumed
ancestorsofchloroplasts
2.Manyoftheproteinsresidingwithinthylakoidmembraneareencodedbychloroplastgenes&
synthesizedonmembraneboundribosomesofthechloroplast

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THE HUMAN PERSPECTIVE: DISORDERS RESULTING FROM

DEFECTS IN LYSOSOMAL FUNCTION
I.Firstdiscoveryofmechanismfortargetingproteinstoparticularorganelleswasdiscoverythatmannose6
phosphateresiduesinlysosomalenzymesactasanaddressfordeliveryoftheseproteinstolysosomes
A.Discoveryoflysosomeaddresswasmadeinstudiesofpatientswitharare&fatalinheritedcondition(I
celldisease)
B.Manycellscontainbloatedlysosomesthatarebloatedwithundegradedmaterials;thishappensbecauseof
theabsenceofhydrolyticenzymes
C.Whenfibroblastsfromthesepatientswerestudiedinculture,itwasfoundthatlysosomalenzymesare
synthesizedatnormallevelsbuttheyaresecretedintomedium&nottargetedtolysosomes
D.Furtheranalysisshowedthatthesecretedenzymeslackedmannosephosphateresiduespresentonthe
correspondingenzymesofcellsfromnormalindividuals
E.TheIcelldefectwassoontracedtoadeficiencyofanenzyme(Nacetylglucosaminephosphotransferase)
requiredformannosephosphorylation
II.H.G.Hers(Univ.ofLouvaininBelgium,1965)explainedhowabsenceofseeminglyunimportant
lysosomalenzyme(glucosidase)leadstodevelopmentoffatalinheritedcondition(Pompedisease)
A.Hesuggestedthatinabsenceofglucosidase,undigestedglycogenaccumulatedinlysosomes,causing
swellingoforganelles&irreversibledamagetocells&tissues
B.Diseasesofthistype,characterizedbydeficiencyofasinglelysosomalenzyme&thecorresponding
accumulationofundegradedsubstrate,arecalledlysosomalstoragedisorders
1.>40ofthesehavebeendescribed,affecting~8,000infants;theirsymptomscanrangefromverysevere
tobarelydetectable,dependingprimarilyonthedegreeofenzymedysfunction
2.Severaldiseaseshavealsobeentracedtomutationsinlysosomalmembraneproteinsthatimpairthe
transportofsubstancestothecytosol
III.AmongthebeststudiedlysosomalstoragedisordersisTaySachsdisease,whichresultsfromadeficiency
oftheenzymeNhexosaminidaseA,anenzymethatdegradesthegangliosideG M2
A.GM2isamajorcomponentofbraincellmembranes
1.Intheabsenceofhydrolyticenzyme,gangliosideaccumulatesinbraincellcytoplasmcausinga
dysfunction
2.Initssevereform,whichstrikesduringinfancy,thediseaseischaracterizedbyprogressivemental&
motorretardation,aswellasskeletal,cardiac,&respiratoryabnormalities
B.Thediseaseisveryrareinthegeneralpopulationbutreachesanincidenceupto1in3600newborns
amongJewsofeasternEuropeanancestry
1.Diseaseincidencehasdroppeddramaticallyinthisethnicpopulationrecentlyasaresultofthecarrier
identification,geneticcounselingofparentsatrisk&prenataldiagnosisbyamniocentesis
2.Allofthelysosomalstoragedisorderscanbediagnosedprenatally
IV.Recently,prospectsfortreatmentoflysosomalstoragedisordershaveimprovedwithdemonstrationthatthe
symptomsofGaucher'sdiseasecanbealleviatedbyenzymereplacementtherapy
A.Gaucher'sdiseaseisadeficiencyofthelysosomalenzymeglucocerebrosidase
B.InfantswithGaucher'sdiseaseaccumulatelargequantitiesofglucocerebrosidelipidsinmacrophage
lysosomes,causingliver&spleenenlargement&anemia
C.Initialattemptstocorrectthediseasebyinfusingasolutionofnormalhumanenzymeintobloodstream
wereunsuccessfulsinceenzymewastakenupbylivercells(notseriouslyaffectedbythedeficiency)

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D.Totargetmacrophages,enzymewaspurifiedfromhumanplacentaltissue&treatedwith3different
glycosidasestoremoveterminalsugarsontheenzyme'soligosaccharidechains
1.Thisexposedtheunderlyingmannoseresidues
2.Afterinfusionintobloodstream,thismodifiedenzyme(marketedundernameCeredase)orthenewer
recombinantenzymeCerezymeisrecognizedbymannosereceptorsonmacrophagesurface
3.Theyarethenrapidlytakenupbyreceptormediatedendocytosis
4.Sincelysosomesarenaturaltargetsiteofmaterialsbroughtintomacrophagebyendocytosis,the
enzymesareefficientlydeliveredtotheprecisesitesinthecellwherethedeficiencyismanifested
5.Thousandshavebeensuccessfullytreatedinthisway
E.Clinicaltrialsofenzymereplacementtherapyfortreatmentofseveralotherlysosomalstoragedisorders
arealsoshowingpromisingresults
1.Unfortunately,manyofthesediseasesaffectthecentralnervoussystem,whichisunabletotakeup
circulatingenzymesbecauseofthebloodbrainbarrier
F.Alternateapproachhasshownsomepromiseinpreclinicaltrials;calledsubstratereductiontherapy,init
smallMWdrugsareadministeredtoinhibitsynthesisofsubstancesthataccumulateinthedisease
G.Finally,althoughitleadstoconsiderablerisktothepatient,bonemarrow(orcordblood)transplantation
hasprovenrelativelysuccessfulintreatingsomeofthesediseases
1.Itisthoughtthattheforeigntransplantedcells,whichcontainnormalcopiesofthegeneinquestion,
secretealimitedamountofthenormallysosomalenzyme
2.Someoftheseenzymemoleculesarethentakenupbythepatient'sowncells,whichlessensthe
impactoftheenzymedeficiency

EXPERIMENTAL PATHWAYS: RECEPTOR-MEDIATED

ENDOCYTOSIS
I.Embryonicdevelopmentbeginswhensmallsperm&muchlargeregg(developsfromoocyte,which
accumulatesyolkmadeelsewhereinfemale'sbody)fusehowdohighMWyolkproteinsenteroocyte?
A.ThomasRoth&KeithPorter(Harvard,1964)reportedonmechanismbywhichyolkproteinsmightbe
takenintomosquitooocytes
1.Duringstagesofrapidoocytegrowth,therewasdramaticincreaseinthenumberofpitlikedepressions
seenonoocytesurface
2.Thepits,formedbyinvaginationofoocyteplasmamembrane,werecoveredontheirinnersurfaceby
abristlycoat
3.Roth&Porterpostulatedthatyolkproteinswerespecificallyadsorbedontotheoutersurfaceofthe
membranesofthecoatedpits,whichwouldtheninvaginateascoatedvesicles
4.Thecoatedvesicleswouldlosetheirbristlycoat&fusewithoneanothertoproducethelarge,
membraneboundyolkbodiescharacteristicofthematureoocyte
B.TokuKanaseki&KenKadora(Univ.ofOsaka,1969)providedinsightintostructureofcoatedvesicles;
didEMexaminationofcrudevesiclefractionisolatedfromguineapigbrains
1.Coatedvesicleswerecoveredbyapolygonalbasketwork
2.Suggestedthatcoatingswereanapparatustocontroltheinfoldingofplasmamembraneduringvesicle
formation
II.BarbaraPearse(MedicalResearchCouncil,Cambridge,England,1975)firststudiesofbiochemicalnature
ofthevesiclecoat
A.Developedprocedureinwhichmembranevesiclesfrompigbrainswerecentrifugedthroughasuccession
ofsucrosedensitygradientsuntilapurifiedfractionofcoatedvesicleswasobtained

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B.Proteinfromcoatedvesicleswassolubilized&fractionatedbySDSPAGE;thecoatcotainedone
predominantproteinspecieswithamolecularmassof~180,000daltons;calledtheproteinclathrin
C.Foundthesameprotein(basedonmolecularmass&peptidemapping)inpreparationsofcoatedvesicles
thatwereisolatedfromseveraldifferenttypesofcellsobtainedfrom>1animalspecies
III.MichaelBrown&JosephGoldstein(Univ.ofTexasMed.Sch.DallasTX;1973)independentlineof
research;interestedininheritedconditionfamilialhypercholesterolemia(FH)
A.Peoplehomozygousfordefectivegene(FHallele)hadprofoundlyelevatedserumcholesterollevels(800
mg/dlvs.200mg/dlforanormalperson)
1.Theyinvariablydevelopedseverelyblocked(atherosclerotic)arteries&usuallydiedfromheartattack
beforetheageof20
2.Atthattime,verylittlewasknownaboutthefundamentalphysiologicorbiochemicaldefectsinthe
disorder
B.Beganstudiesbyexaminingcholesterolmetabolisminculturedfibroblastsderivedfromtheskinof
normal&FHafflictedindividuals
1.Foundthatratecontrollingenzymeincholesterolbiosynthesis,HMGCoAreductase,couldbe
inhibitedinnormalfibroblastsbycholesterolcontaininglipoproteins(likeLDL)placedinmedium
2.LDLadditiontoculturemediuminwhichnormalfibroblastsweregrowingledtodecreasedlevelof
HMGCoAreductaseactivity&correspondingdecreaseincholesterolsynthesisbyfibroblasts
3.WhenHMGCoAreductaselevelsweremeasuredinFHderivedfibroblasts,theywerefoundtobe40
60timesthatofnormalfibroblasts
4.Also,enzymeactivityinFHfibroblastswastotallyunaffectedbypresenceofLDLinmedium
C.Howcouldlipoproteininmediumaffectactivityofenzymeincytoplasmofculturedcells?Brown&
Goldsteininitiatedstudiesontheinteractionbetweenthecells&thelipoproteins
1.AddedradioactivelylabeledLDLtoculturedishescontainingasinglelayeroffibroblastsderived
fromeitherFHafflictedornormalhumansubjects
2.ThenormalfibroblastsboundthelabeledLDLmoleculeswithhighaffinity&specificity,butthe
mutantcellsshowedvirtuallynoabilitytobindtheselipoproteinmolecules
3.ThisindicatedthatnormalcellshaveahighlyspecificreceptorforLDL&thatthereceptorwas
defectiveormissingincellsfrompatientswithFH
D.Brown&GoldsteinteamedupwithRichardAndersonwhowasstudyingcellstructurewiththeEM
1.Theyincubatedfibroblastsfromnormal&FHsubjectswithLDLthathadbeencovalentlylinkedto
theironcontainingproteinferritin,whichbecauseofironcontentcanscatteranelectronbeam
2.ThusitcanbevisualizedinEM
3.WhennormalfibroblastswereincubatedwithLDLferritinat4C,atemperatureatwhichligandscan
bindtocellsurfacebutcannot beinternalized,LDLferritinparticleswereboundtocellsurface
4.TheLDLparticleswerenotrandomlyscatteredoverthecellsurface,butwerelocalizedtoshort
segmentsofplasmamembranewherethemembranewasindented&coatedbyafuzzymaterial
5.ThesesegmentsofmembraneweresimilartothecoatedpitsdescribedbyRoth&Porter&hadsince
beenseeninavarietyofcelltypes
6.CellsfromFHpatientshadasimilarnumberofcoatedpitsonsurfacebutnoLDLferritinwasbound
tothesemutantcells
7.ConcludedthatmutantFHalleleencodedareceptorthatwasunabletobindLDL
8.SubsequentEMstudiesonLDLferritininternaliztionrevealedtheendocyticpathwaybywhichthese
lipoproteinparticleswereinternalized
E.TheypostulatedthatrapidinternalizationofreceptorboundLDLisstrictlydependentonlocalizationof
LDLreceptorsincoatedpits

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1.IfLDLreceptorfailedtobelocalizedwithincoatedpit,itwouldnotbeabletodeliveritsbound
ligandtocellularlysosomes&thuswouldnotbeabletoaffectcholesterolbiosynthesiswithincell
IV.AnLDLreceptorwithadifferentkindofmutationwassoonfound
A.Thisdefect(knownasJ.D.mutationafterthepatientinwhichitoccurred)boundnormalamountsof
radioactivelylabeledLDL,yetreceptorboundLDLfailedtobeinternalized
1.Thus,itwasnotdeliveredtocytoplasmiclysosomesforprocessing
B.Andersonetal.postulatedthatLDLreceptorwastransmembraneproteinthatnormallywaslocalizedin
coatedpits,becauseitscytoplasmicdomainwasspecificallyboundbycoatedpitcomponent
1.Thecomponentwasthoughttopossiblybeclathrinbutwaslateridentifiedasasalikelysubunitofan
APadaptor
2.Becauseofadefectinitscytoplasmicdomain,theJ.D.mutantreceptorwasunabletolocalizeina
cell'scoatedpits
3.PeoplewiththismutationexhibitsamephenotypeaspatientswhosereceptorscannotbindLDL
C.LaterstudiesshowedthatnormalLDLreceptoris839aminoacidtransmembraneglycoprotein,with50
aminoacidsatCterminalendofproteinextendinginwardfrommembraneascytoplasmicdomain
1.Theproteincontainedasingleaminoacidsubstitution;atyrosineresiduenormallylocatedatposition
807wasreplacedbyacysteine
2.Thissinglechangeinaminoacidsequenceobliteratedprotein'sabilitytoconcentrateincoatedpits
D.Attentionturnedtotheaminoacidsequencesofthecytoplasmictailsofotherreceptorsthatgetlocalized
tocoatedpitswerethereanycommoninternalizationsignals?2suchsignalsturnedup
1.Bothcontainedtyrosine(Yinsingleletternomenclature):alesscommonNPXYsignal(asintheLDL
receptor)&YXXsignal(asinthetransferrinreceptor)
2.IntheYXXsignal,Xcanbeanyaminoacid&isanaminoacidwithabulky,hydrophobicside
chain
3.TheYXXsequenceofthereceptorbindstothesubunitoftheAP2adaptors;Xraycrystallography
hasrevealedthenatureofinteractionbetweenadaptor&internalizationsignal
4.Meanwhile,theAP2adaptorcomplexbindstotheclathrincoatbymeansofitssubunit
5.Asaresultofthesevariousintermolecularcontacts,theadaptorcomplex&receptoraretrappedin
coatedpitspriortoendocytosis
V.Linesofinvestigationaboutclathrinmediatedendocytosisfollowedoverthepastdecade
A.2componentsofendocyticmachineryhavebeenlabeledwithdifferentfluorescentmarkers&their
movementswerefollowedovertimewithinalivingcell
1.Dynamin,AP2adaptorsorvarioustypesofcargoreceptorshavebeenseentobindtoclathrincoated
pits&getenvelopedinclathrincoatedvesicle,whichbudsoffintocytoplasm&disappears
B.Engineerculturedmammalianepithelialcellstoexpressaclathrinlightchainthatisfusedtoavariantof
thegreenfluorescentprotein
1.Whenobservedbyfluorescencemicroscopy,seegreenpatchesoncellsurface,whichforthemostpart
representclathrincoatedpitssituatedatthecellsurface
2.RedstainingspotsonslideareindividualfluorescentlylabeledLDLparticlesthatwereaddedtothe
mediuminwhichthecellsweregrowing
3.OnceLDLparticlehasbeenboundtocoatedpitLDLreceptor,theoverlapofthetwofluorescentdyes
producesayelloworangespot
4.LaterseeuncoatedvesiclewithcontainingredfluorescentLDLparticlesmovingintotheadjacent
cytoplasm

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QUESTES
1.

OmRNAdeumaproteinsecretorabemconhecidafoiisoladoecolocadonumtubodeensaio,
juntamentecomoutrassubstnciasnecessriassnteseproteicainvitro.Asequnciadaprotena
produzidainvitroeradiferentedasequnciadaprotenasecretorapurificada.Qualapossvelexplicao
paraestefacto?

2.

Submeteramseclulasemcrescimentoaumpulsobrevecomradioactividade,tornadoradioactivosos
fosfolpidos.Algumtempodepois,observouseradioactividadeprimeiramentesobreoretculo
endoplasmtico.Posteriormente,aparecenamembranacelular,logoapstersidoobservadasobreo
complexodeGolgieemvesculascitoplasmticas.Aqueconclusesapontamosdadosapresentados?

3.

UmamolculachamadaaequorinaproduzfluorescnciaquandoseligaaiesCa2+.Seestiveraobservar
umaclulanaqualseinjectouaequorinaeseessaclulasegregardeterminadasprotenas,oquese
observaimediatamenteantesdasecreodessasprotenas?

4.

SeumaamebafortratadacomcitocalasinaB,uminibidordapolimerizaodaactina,oprocessode
fagocitosepra.Qualrazoseissoacontecer?

5.

Umamulherdeuluzumbebincapazdeadquiriradequadamenteosanticorposprovenientesdoleite
materno.Posteriormente,determinousequeeleproduziareceptoresparaosanticorposequeestesse
ligavamaessesreceptores.Indiqueumapossvelrazoparaaincapacidadedobebadquiriros
anticorposmaternos!

6.

Qualacausadasilicoseedaasbestose?

7.

Porquerazoasenzimaslisossomaiscausamartritereumatidequandosolibertadasparaoespao
extracelular?

8.

ExpliqueporquequeoslisossomasficaminchadosnospacientesquesofremdadoenadasclulasI!

9.

Qualadoenacaracterizadapelaausnciaoumalformaodaenzimaglucosidase?Qualopapel
fisiolgicodaenzimaglucosidase?

10. Nafigura8.2b,oqueaconteceriataxacomqueaviaconstitutivaocorreseaviasecretoraregulada
recebesseestmulosquereduzissemataxadesecresso?

11. Qualafunodascisternasachatadaseempilhadasdoretculoendoplasmticorugoso,observadasna
figura8.9ad?
12. Relativamentefigura8.11a:
a) Quaisosorganeloslocalizadosnapartebasaldaclula?

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b) Quaisosorganeloslocalizadosnaparteapicaldaclula?
c) OndeseencontraocomplexdeGolgi?
13. Nafigura8.13,oqueimpedeacadeiapolipeptdicadeumaprotenamembranarintegralqueestaser
formadadepassarcompletamenteatravsdotransloco?
14. Afigura8.14representaaadioassimtricadegruposdeoligossacardeosaprotenas.Seumadada
protenaintegraltiveralgunsacaresnoladocitoplasmticodamembranaplasmtica,emqualdas
extremidadesdessaprotenaterosidoadicionadososacares?
15. Deacordocomafigura8.16,emqualdassuperfciesdoRetculoEndoplasmticosoadicionadosos
primeirosacaresaofosfatodedolicoledequecompartimentosoobtidososltimosacaresaserem
adicionados?
16. Deacordocomafigura8.27,oqueaconteceriasefosseadicionadoGTPemexcessorelativamentoao
seuanlogoGTPS?
17. Afigura8.29bmostraasequnciadeeventosenvolvidosnodireccionamentodeenzimaslisossomais
paraoslisossomas.Oqueaconteceriasefossebloqueadaafosforilaoderesduosdemanosedas
enzimaslisossomais?
18. Deacordocomafigura8.29,seumapessoativesseumaalteraogenticaqueresultasseemreceptores
deenzimaslisossomaisdeficientes,quaisseriamosefeitosfisiolgicosdestaalterao?Estapessoateria
umproblemamdico?
19. DequemodoserelacionamosnveisdeLDLcomosataquescardacos?

103