ProteinSorting

intheCell
Eukaryoticcellscytoplasmcontain
manydifferentspecialized
compartmentscalled.. organelles
Eachorganellecontainsadistinctsetofproteins
thatmediatesitsuniquefunctions.
Proteinswithineukaryotesare
sortedbytwo basictypesofprotein
targetingpathways
PROTEINTARGETINGANDSORTING
Posttranslational pathway targets newly synthesized
proteins to their appropriate compartment. This pathway is
utilized by proteins destined to the nucleus, mitochondria,
chloroplastandperoxisome.
Cotranslational pathway involves the cotranslational
transport ofaproteinintotheendoplasmicreticulumlumen.
ThispathwayisutilizedbyproteinsdestinedfortheER,Golgi
apparatus, lysosome, or the plasma membrane. Secreted
proteinsarealsoproducedandtargetedbythismechanism.
Aftertranslation,
proteinsmovetovarious
compartmentby:
(i)gatedtransport
(ii)transmembrane
transport,
(iii)vesiculartransport.
KeyelementsfortheProteintargetingevents
Signalsequences
Shortregionsofaproteinthatactastargetingsignals
todirecttheproteintospecificsubcellular localization
Receptors thatrecognizeparticularsignalsequences
Translocationchannelsthatallowtransferof
proteinsacrossthemembranebilayer.
Requireenergy (ATPorGTP)
Examplesofdifferenttypesofsignalsequence
Thesesequencesfunctionlikecellular
PostalCodestargetingproteinstowards
theirdestinationinthecell.
(A)SignalsequencesmaybepresentinNterminalor
(B) distributedwithintheproteinsequencewhichfoldto
formaSIGNALPATCH.
TheTransportof
Moleculesinto
andoutofthe
Nucleus
Manyproteinsthatfunctioninthenucleus
includinghistones,DNAandRNA
polymerases,generegulatoryproteins,and
RNAprocessingproteinsareselectively
importedintothenuclearcompartment
fromthecytosol.
Atthesametime,tRNAs andmRNAs
aresynthesizedinthenuclear
compartmentandthenexportedto
thecytosol.
Nucleartransportinvolvesthepassagethrougha
gate thatseparatestwoaqueouscompartments,
thecytoplasmandthenucleoplasm.
Smallmolecules(5000daltons orless)can
diffusefreelythroughthenuclearpore,
largermoleculesrequireactivetransport.
Largerproteinsbindtospecificreceptorproteins
locatedintheporecomplexesandarethenactively
transportedacrossthenuclearenvelopethrough
thecomplexes.
Thenuclearenvelopeofatypicalmammaliancell
contains3000to4000nuclearporecomplexes.
IfthecellissynthesizingDNA,itneedsto
importabout10
6
histone moleculesfrom
thecytosol every3mininorderto
packagenewlymadeDNAintochromatin.
Key element for nuclear transport
1. Nuclear Localization Sequence (NLS)
2. Nuclear pores complex
3. Transport protein
(i) Importins: Cytoplasm to nucleus
(ii) Exportins: Nucleus to cytoplasm
Forproteinstoolargetodiffusethroughthe
NuclearPore,anNuclearLocalizationSequence
(48aa length)isrequiredfornuclearimport
NuclearTransport:CytoplasmtoNucleus
Step1.ProteincontainingNLSbindssolubleNLSreceptor
importin atthecytoplasmic side
Step2 Importin:NLS proteincomplextransportstocytoplasmic
filaments
Step 3. Cytoplasmic filaments bend toward nucleus
Step 4. Change in conformation of transporter
Step 5. Inside the nucleus, Importin:NLS protein
complex interaction with Ran-GTP causes
a conformational change in the importin that causes
dissociation
Nuclearexportroughlyreversestheimportprocess;inthenucleus,theExportin
bindsthecargoandRanGTPanddiffusesthroughtheporetothecytoplasm,
wherethecomplexdissociates.
NuclearTransport:NucleustoCytoplasm
TheGTPbindingproteinRanregulatesNuclearTransport
EXPORT
IMPORT
Importins
Exportins
Protein
withNLS
Protein
withNES
TheRanGTP/GDPcycle
RanGAPRanGTPaseacceleratingprotein
RanGEFRanguaninenucleotideexchangefactor
TheTransportof
Proteinsinto
Mitochondriaand
Chloroplasts
Mitochondriaandchloroplastsaredouble
membrane enclosedorganelles.
BothorganellescontaintheirownDNA,
ribosomes,andothercomponents
requiredforproteinsynthesis,mostof
theirproteinsareencodedinthecell
nucleusandimportedfromthecytosol.
There are two subcompartments in mitochondria: the
internalmatrixspaceandtheintermembrane space.
Chloroplasts have the same two subcompartments plus
an additional subcompartment, the thylakoid space,
which is surrounded by the thylakoid membrane. Each
of the subcompartments in mitochondria and
chloroplastscontainsadistinctsetofproteins.
Mostofthemitochondrial
precursorproteinshaveasignal
sequenceattheirNterminusthat
israpidlyremovedafterimportby
aprotease(thesignalpeptidase)in
themitochondrialmatrix.
Whenthesignalsequenceisfolded
asan helix,thepositively
chargedresidues(red)areseento
beclusteredononefaceofthe
helix,whilethenonpolar residues
(yellow)areclusteredprimarilyon
theoppositeface.
Proteintranslocationfromthecytoplasmtothe
mitochondrialmatrixrequirestwodistincttranslocation
complexesTom&Tim:theTOMcomplexfunctionsacross
theoutermembrane,andtwoTIMcomplexes,theTIM23
andTIM22complexes,functionacrosstheinner
membrane.
Cytoplasmic proteinscrossboththeouterandinnermitochondrial
membranesinasinglestep:
TOMcomplex:Itrecognizesthesignalsequencesofallnucleusencoded
mitochondrialproteinsandimportintotheintermembrane space.
TIM23complex:Ittransportsproteinsintothematrixspace.
TIM22complex:Itmediatestheinsertionofasubclassofinner
membraneproteins,includingthecarrierproteinthattransports ADP,
ATP,andphosphate.
OXAcomplex:Itmediatestheinsertionofinnermembraneproteinsthat
aresynthesizedwithinthemitochondriaintheinnermitochondrial
membrane.Italsohelpstoinsertsomeproteinsthatareinitially
transportedintothematrixbytheTOMandTIMcomplexes.
ATPhydrolysisandaH+gradientareusedtodrive
proteinimportintomitochondria.
Proteinimportintothemitochondrialmatrixisastepwiseprocess:
1)Bound cytosolic hsp70 isreleasedfromthe protein inastepthatdependsonATP
hydrolysis.Afterinitialinsertionofthe signalsequence andofadjacentportionsofthe
polypeptide chainintothe TOMcomplex,thesignalsequenceinteractswithaTIM
complex.
2)Thesignalsequenceisthentranslocated intothematrixinaprocessthatrequiresan
electrochemical H
+
gradient acrossthe innermembrane,positioningtheunfolded
polypeptidechainsothatittransientlyspansbothmembranes.
3)Mitochondrialhsp70 bindstoregionsofthepolypeptidechainastheybecomeexposed
inthematrix,therebypulling theproteinintothematrix.ATPhydrolysisthenremoves
themitochondrialhsp70,allowingtheimportedproteintofold.
Proteintransportintochloroplasts
Itresemblestransportintomitochondriainmanyrespects.
Bothprocessesoccurposttranslationally,useseparate
translocationcomplexesineachmembrane,occuratcontact
sites,requireenergy,anduseNterminalsignalsequences.
Proteinsaretransportedfromthecytosol totheirfinal
destinationintwosteps.
1.First,theypassacrossthedoublemembraneatcontactsites
intothematrixspaceofthechloroplast,calledthestroma,
and
2.Secondly,theyaretranslocated intothethylakoid
membrane(oracrossthismembraneintothethylakoid space)
Translocationintothethylakoid spaceorthylakoid
membranecanoccurbyanyoneofatleastfour
routes:
1.Secpathway,
2.SRP(signalrecognitionparticle)likepathway,
3.pH pathway,
4.Spontaneousinsertionpathway
TheTransport
ofProteins
into
Peroxisome
Peroxisomes
Cytoplasmic organelles
Boundedbyasinglemembrane.
Usedbythecellasaplaceto
sequesterspecificreactions.
Theseoftengeneratehydrogen
peroxide
Importofperoxisomal matrixproteins
isposttranslational
Onewelldefinedperoxisomal import
signalisthesequenceSerLysLeu at
theextremeCterminusofaprotein.
Mechanismofimportisnotwell
defined.
Transportintothe
EndoplasmicReticulum
(Gatewaytothe
Secretory Pathway)
TheRough EndoplasmicReticulumwith
ribosomes attachedisthesiteofcotranslational
translocation ofproteinsintotheER
Incotranslationaltranslocation,thenascent
proteincrossestheERmembraneasitleaves
theribosome
ERtargetingsignalsarerecognizedbySRP
(SignalRecognitionParticle)
SRPrecognizessignalsequencesastheycomeoffthe
ribosomeandcarriesthemRNAribosomenascent
polypeptidecomplextotheER
SRPcomplexthenbindstoSRPreceptor.Bindingto
receptorresultstranslocationofpolypeptidetoER
lumenwhereastranslationcontinues.SRPandSRP
receptorlaterdissociatesandstartfreshcycle.
TranslocationofasolubleproteinintotheERlumen
Integralmembraneproteinscontainstoptransfer
sequencesthatleadtoreleaseoftheproteinintothe
ERmembrane
Onceproteinshaveenteredtheendoplasmic
reticulumtheycanbemoveontoother
compartmentsofthesecretory pathwayorout
ofthecellentirely.
Butinadditiontoactingasaportofentryfor
proteinsintothesecretory pathway,two
additionalimportantfunctionsforprotein
targetingtakeplaceintheER.
Nlinkedglycosylation ofproteins
Proteinfolding
TheEndoplasmicReticulum
isthegatewayforprotein
transportintoalltheother
membraneboundorganelles
ofthesecretory pathway.
Proteinsandlipidsare
traffickedthroughthe
secretory pathway
insmallcarrierscalled
vesicles.
TheSecretory Pathway
lysosome
Transportfromthe
ERthroughthe
Golgi Apparatus
ProteinsthathaveenteredtheERand
aredestinedfortheGolgi apparatusor
beyondarefirstpackagedintosmall
COPIIcoated transportvesicles.These
transportvesiclesbudfromspecialized
regionsoftheERcalledERexitsites,
whosemembranelacksbound
ribosomes.
TheStepsofVesicleTransport
Aftertransportvesicleshavebuddedfrom
anERexitsiteandhaveshedtheircoat,
theybegintofusewithoneanother
formingvesiculartubularclusters.
Membranefusionrequiresasetof
matchingSNAREs [SNAP
(Soluble NSF Attachment
Protein) Receptor]
Vesicle Fusionrequires SNAREproteins
Oncethevesiculartubularclustersareform,theybeginto
buddingoffresultingintonewvesicles,thesevesiclesare
COPIcoated.TheycarrybackERresidentproteinsthathave
escapedaswellasproteinsthatparticipatedintheERbudding
reaction.
Formationofatransportvesiclerequirescoatproteins
EachGolgi stackhastwodistinct
faces:acis face(orentryface)anda
transface(orexitface).
Bothcis andtransfacesareclosely
associatedwithspecial
compartments,eachcomposedofa
networkofinterconnectedtubular
andcisternal structures:thecis Golgi
network(CGN)(alsocalledthe
intermediatecompartment)andthe
transGolgi network(TGN),
respectively.
ProteinsexitingfromtheTGNcaneither
moveonwardandbesortedaccordingto
whethertheyaredestinedforlysosomes,
secretory vesicles,orthecellsurface.
Transportfrom
theTransGolgi
Networkto
Lysosomes
Lysosomes aremembrane
enclosedcompartmentsfilledwith
hydrolyticenzymesthatareused
forthecontrolledintracellular
digestionofmacromolecules.
Allareacidhydrolases.Foroptimal
activitytheyrequireanacid
environment,andthelysosome
providesthisbymaintainingapH
ofabout5.0initsinterior.AnH
+
pumpinthelysosomal membrane
usestheenergyofATPhydrolysis
topumpH
+
intothelysosome,
therebymaintainingthelumenat
itsacidicpH.
Newlysynthesizedlysosomal proteinsaretransferredinto
thelumenoftheER,transportedthroughtheGolgi
apparatus,andthencarriedfromthetransGolgi networkto
lateendosomes bymeansofclathrincoatedtransport
vesicles.
Thelysosomal hydrolases containNlinkedoligosaccharides
thatarecovalentlymodifiedinauniquewayinthecis Golgi
network
Thesemannose6phosphate(M6P)groupsarerecognized
byanM6PreceptorproteininthetransGolgi networkthat
segregatesthehydrolases andhelpspackagetheminto
buddingtransportvesiclesthatdelivertheircontentsto
lateendosomes.TheM6Preceptorsshuttlebackandforth
betweenthetransGolgi networkandtheseendosomes.
TransportintotheCell
fromthePlasma
Membrane:Endocytosis
Endocytosis isaprocessinwhichtheparticlemoveinward
fromthecellsurfacetolysosomes.
Twomaintypesofendocytosis aredistinguishedonthebasis
ofthesizeoftheendocytic vesiclesformed.
Phagocytosis (cellulareating),whichinvolvestheingestion
oflargeparticles,suchasmicroorganismsordeadcellsvia
largevesiclescalledphagosomes (generally>250nmin
diameter).Itismainlycarriedoutbyspecializedcells
macrophages,neutrophils,anddendritic cells.
Pinocytosis(cellular drinking),whichinvolvestheingestionof
fluidandsolutesviasmallpinocytic vesicles(about100nmin
diameter).Pinocytic vesiclesoftenbeginsatclathrincoatedpits
intheplasmamembrane
lysosome
Mosteukaryoticcellsarecontinually
ingestingfluidandsolutesbypinocytosis;
largeparticlesaremostefficientlyingested
byspecializedphagocytic cells.Pinocytosis,
whichisaconstitutiveprocessthatoccurs
continuously.