mueckler review slides - washington university in st. louis sessions/2017 review... ·...
TRANSCRIPT
MCBTest1– MuecklerReview
9-30-17
Func%onsofCellularMembranes1. Plasmamembraneactsasaselec%velypermeablebarriertothe
environment• Uptakeofnutrients• Wastedisposal• Maintainsintracellularionicmilieu2. Plasmamembranefacilitatescommunica%on• Withtheenvironment• Withothercells3.Intracellularmembranesallowcompartmentaliza%onandsepara%onofdifferentchemicalreac%onpathways• Increasedefficiencythroughproximity• PreventfuHlecyclingthroughseparaHon• ProteinsecreHon
FaJyAcidSaturaHon
Figure 10-11a Molecular Biology of the Cell (© Garland Science 2008)
(Hydrophobic)
(Hydrophilic)
BilayersareThermodynamicallyStableStructuresFormedfromAmphathicLipids
Figure 10-8 Molecular Biology of the Cell (© Garland Science 2008)
TheForma%onofCell-LikeSphericalWater-FilledBilayersisEnerge%callyFavorable
Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008)
PhosphoLipidMovementswithinBilayers(µM/sec)
(1012-1013/sec) (108-109/sec)
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)
A“Scramblase”EnzymeCatalyzesSymmetric
GrowthofBothLeafletsintheER
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)
A“Flippase”EnzymepromotesLipid
AsymmetryinthePlasmaMembrane
Figure 10-17a Molecular Biology of the Cell (© Garland Science 2008)
PhospholipidsareInvolvedinSignalTransduc%on
PI-4,5P PI-3,4,5P
1.Ac%va%onofLipidKinases
2.PhospholipasesProduceSignalingMoleculesviatheDegrada%onof
Phospholipids
CholesterolBiosynthesisOccursintheCytosolandattheERMembraneThroughIsoprenoidIntermediates
(Rate-LimiHngStepinER)
3WaysinwhichLipidsMaybeTransferredBetweenDifferentIntracellularCompartments
VesicleFusionDirectProtein-Mediated
TransferSolubleLipid
BindingProteins
Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)
The3BasicCategoriesofMembraneProtein
Integral Lipid-Anchored
Peripheral(canalsointeractviaPLheadgroups)
Single-Pass Mul%-pass
Fa_yacylanchor
GPIAnchor
TransmembraneHelix
LinkerDomain
β-Strands
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)
MembraneDomainsare“Inside-Out”
Right-SideOutSolubleProtein
Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008)
Func%onalCharacteriza%onofIntegralMembraneProteinsRequiresSolubiliza%onandSubsequentRecons%tu%onintoaLipidBilayer
Figure 10-39 Molecular Biology of the Cell (© Garland Science 2008)
4WaysthatProteinMobilityisRestrictedinBiologicalMembranes
IntramembraneProtein-ProteinInteracHons
InteracHonwiththecytoskeleton
InteracHonwiththeextracellularmaxtrix
IntercellularProtein-ProteinInteracHons
Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)
RibosomalSubunitsareShared
BetweenFreeandMembrane-Bound
Polysomes
Targe&nginforma&onresides
intheNascentpolypep&dechain
Signal-MediatedTarge%ngtotheRER
Proper%esofSecretorySignalSequences
HydrophobicCoreN MatureProtein
8-12Residues
15-30Residues
++
• LocatedatN-terminus• 15-30Residuesinlength• Hydrophobiccoreof8-12residues• OZenbasicresiduesatN-terminus(Arg,Lys)• Nosequencesimilarity
cleavage
InVitroTransla%on/Transloca%onSystem
• mRNA• Roughmicrosomes• Ribosomes• tRNAs• SolubletranslaHonfactors• LowMWcomponents• Energy(ATP,creaHne-P,creaHnekinase)
ReHculocyteorwheatgermlysate
InVitroTransla%on/Transloca%onSystem
mRNA+
TranslaHonComponents
+Aminoacid*
Protein* SDSPAGE
InVitroTransla%onofProlac%nmRNA
ProlacHnisapolypepHdehormone(MW~22kd)secretedbyanteriorpituitary
12345678MW(kd)
2522
Lanes:1. PurifiedprolacHn2. NoRM3. RM4. NoRM/digestwith
Protease5. RM/digestwith
Protease6. RM/detergenttreatand
addProtease7. ProlacHnmRNAminus
SS+RM/digestwithProtease
8. SS-globinmRNA+RM/digestwithProtease
18
SDSGel
Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008)
Mul%passTopologiesareGeneratedbyMul%pleInternalSignal/AnchorSequences
TypeIVa
+–
+–
+–
+–
TheChargeDifferenceRuleforMul%spanningMembraneProteins
NH2 COOH
NH2
COOH
+ + –
+ +
NH2 COOH
NH2
COOH
+ +
+ +cytoplasm
cytoplasm
––
–
–
–
–
Figure 12-53 Molecular Biology of the Cell (© Garland Science 2008)
OligosaccharideProcessingintheRERisUsedforQualityControl
DisulfideBridgesareFormedintheRERbyProteinDisulfideIsomerase(PDI)
Figure 12-23 Molecular Biology of the Cell (© Garland Science 2008)
ProteinsareIncorporatedIntoMitochondriaViaSeveralDifferentRoutes
Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008)
ProteinImportintotheMatrixRequiresATPHydrolysisandanIntactProtonGradient
AcrosstheInnerMembrane
Targe%ngtotheInnerMembraneOccursVia3Dis%nctRoutes
Oxa1-MediatedStop-Transfer-Mediated Tom70/Tim22/54-Mediated
Mul%-PassProteinsSingle-PassProteins
CytochromeoxidasesubunitCoxVa
ATPSynthaseSubunit9
ADP/ATPAn%porter
CytochromeB2 CytochromecHemeLyase
Targe%ngtotheIntermembranousSpaceOccursViaTwoDis%nctPathways
DirectDeliveryIMSpaceProtease
Figure 12-27 Molecular Biology of the Cell (© Garland Science 2008)
Targe%ngtotheOuterMembraneViatheSAMProteinComplex
(SorHngandAssemblyMachinery)
(β-Barrell)
Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)
NuclearTransport
• Bidirec%onal• SingleLargePoreComplexSpans2lipidbilayers• NuclearPoresmuchlargerthanothertranslocons
Figure 12-13 Molecular Biology of the Cell (© Garland Science 2008)
NuclearImportandExportSequencesareRecognizedbyDifferentMembersoftheSameReceptorFamily(Keryopherins)
Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008)
NuclearImportandExportOperateViaReciprocalUseoftheRan-GDP/GTPConcentra%onGradient