the signal hypothesis and the targeting of nascent...
TRANSCRIPT
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TheSignalHypothesisandtheTargetingofNascent
PolypeptidestotheSecretoryPathway
Tuesday8/30/2018
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Figure 6-63 Molecular Biology of the Cell (© Garland Science 2008)
RibosomeStructure
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Figure 6-76 Molecular Biology of the Cell (© Garland Science 2008)
FormationofPolyribosomes
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IntracellularTargetingofNascentPolypeptides• Defaulttargetingoccurstothecytoplasm• Allotherdestinationsrequireatargetingsequence• Majorsortingstepoccursattheleveloffreeversusmembrane-boundpolysomes
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Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)
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Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)
RibosomalSubunitsareShared
BetweenFreeandMembrane-Bound
Polysomes
Targetinginformationresides
intheNascentpolypeptidechain
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Signal-MediatedTargetingtotheRER
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PropertiesofSecretorySignalSequences
HydrophobicCoreN MatureProtein
8-12Residues
15-30Residues
++
• LocatedatN-terminus• 15-30Residuesinlength• Hydrophobiccoreof8-12residues• OftenbasicresiduesatN-terminus(Arg,Lys)• Nosequencesimilarity
cleavage
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InVitroTranslation/TranslocationSystem
• mRNA• Roughmicrosomes• Ribosomes• tRNAs• Solubletranslationfactors• LowMWcomponents• Energy(ATP,creatine-P,creatinekinase)
Reticulocyteorwheatgermlysate
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Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)
IsolationofRoughMicrosomesbyDensityGradientCentrifugation
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InVitroTranslation/TranslocationSystem
mRNA+
TranslationComponents
+Aminoacid*
Protein* SDSPAGE
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InVitroTranslationofProlactinmRNA
Prolactinisapolypeptidehormone(MW~22kd)secretedbyanteriorpituitary
12345678MW(kd)
2522
Lanes:1. Purifiedprolactin2. NoRM3. RM4. NoRM/digestwith
Protease5. RM/digestwith
Protease6. RM/detergenttreatand
addProtease7. ProlactinmRNAminus
SS+RM/digestwithProtease
8. SS-globinmRNA+RM/digestwithProtease
18
SDSGel
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IdentificationofaSolubleRERTargetingFactor
Lanes:1. Noadditions2. KRM3. KRM/digestwithProtease4. KRM+KClwash5. KRM+KClwash/digest
withProtease
12345MW(kd)
2522
18
RM+
0.5MKCl
Centrifuge
Pellet=KRMSupernate=KClwash
8
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Lanes:1. Noadditions2. KRM3. KRM/digestwithProtease4. KRM+KClwash/digestwith
Protease5. KRM+SRP/digestwithProtease
12345MW(kd)
2522
18
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PurificationoftheSignalRecognitionParticle(SRP)
KClWash SRPHydrophobic
Chromatography
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SubcellularDistributionoftheSignalRecognitionParticle(SRP)
WhereisSRPlocatedwithinthecell?47%ribosomes+polyribosomes15%cytoplasm38%roughendoplasmicreticulumConclusions:
• SRPlikelymovesbetweendifferentsubcellularcompartments• SRPisasolubleparticlethatcanassociatewithmembranesandisnotapermanentmembrane-boundRERreceptor
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Figure 12-39a Molecular Biology of the Cell (© Garland Science 2008)
StructureoftheSignalRecognitionParticle
(7SLRNA)
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Figure 12-39b Molecular Biology of the Cell (© Garland Science 2008)
InteractionsBetweenSRPandtheSignalSequenceandRibosome
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IdentificationofanIntegralMembraneTargetingFactor
Lanes:1. Noadditions2. SRPOnly3. SRP+KRM/digestwith
Protease
123456MW(kd)
2522
8
KRM DigestwithElastase
CentrifugeE-supernate
E-KRMpellet
4. SRP+E-KRM5. SRP+E-Supernate6. SRP+E-KRM+E-
Supernate
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IdentificationofSRPReceptor
Lanes:1. Noadditions2. SRP3. SRP+SRPReceptor
123MW(kd)
2522
8
KRM SRPReceptor
DetergentSolubilize
SRPAffinityColumn
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Figure 12-42 Molecular Biology of the Cell (© Garland Science 2008)
StructureoftheRERTranslocationChannel(Sec61Complex)
Single-Pass
Single-Pass
10TMS
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Figure 12-43 Molecular Biology of the Cell (© Garland Science 2008)
ASingleRibosomeBindstoaSec61Tetramer
(Side-View)
(LumenalView)
(From2-DEMImages)
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Figure 12-44 Molecular Biology of the Cell (© Garland Science 2008)
Post-TranslationalTranslocationisCommoninYeastandBacteria
SecAATPasefunctionslikeapistonpushing~20aa’sintothechannelpercycle
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ClassificationofMembraneProteinTopology
Single-Pass,Bitopic Multipass,Polytopic
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Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008)
GenerationofaTypeISingle-PassTopology
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Figure 12-47 Molecular Biology of the Cell (© Garland Science 2008)
GenerationofTypeIIandTypeIIISinglePassTopologies
TypeII
TypeIII
Post-translationalTranslocation
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Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008)
MultipassTopologiesareGeneratedbyMultipleInternalSignal/AnchorSequences
TypeIVa
+–
+–
+–
+–
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Figure 12-49 Molecular Biology of the Cell (© Garland Science 2008)
MultipassTopologiesareGeneratedbyMultipleInternalSignal/AnchorSequences
TypeIVb
+
–
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TheChargeDifferenceRuleforMultispanningMembraneProteins
NH2 COOH
NH2
COOH
+ + –
+ +
NH2 COOH
NH2
COOH
+ +
+ +cytoplasm
cytoplasm
––
–
–
–
–
-
+
TransmembraneChargeInversionDisruptsLocalMembraneTopologyinMultipassProteins
NH2 COOH+ –– +1 432
NH2 COOH+ –– +1 432
1 2 3 4
NH2 COOH
1
2 3
4
NH2 COOH
cytoplasm
cytoplasm
L1 L2 L3
L1 L2 L3
L1
L2
L3
–
L1
L2
L3
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Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008)
N-LinkedOligosaccharidesareAddedtoNascentPolypeptidesintheLumenoftheRER
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BiosynthesisoftheDolichol-POligosaccharideDonor
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StructureoftheHigh-MannoseCoreOligosaccharide
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ProcessingoftheHigh-MannoseCoreOligosaccharideintheRER
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Figure 12-53 Molecular Biology of the Cell (© Garland Science 2008)
OligosaccharideProcessingintheRERisUsedforQualityControl
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DisulfideBridgesareFormedintheRERbyProteinDisulfideIsomerase(PDI)