CytoplasmchromosomePeriplasmInner membraneOuter membraneOutside the cellBACTERIAL CELLmRNAproteinMIT Biology Department7.012: Introductory Biology - Fall 2004Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette GardelEUKARYOTIC CELLMitochondrionPlasma membraneNucleusEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellNEukaryoticCellBacteriaInsulinGrowth FactorsAntibodiesInsulin ReceptorGrowth FactorReceptorsHistidine synthesisLactaseGlycolysisEnzymesCyclinsToxinLactose ReceptorgalactosidaseFullySecretedProtein(Outside the Cell)MembraneProteinCytoplasmicProteinExamplesGeorge PaladeImages removed due to copyright reasons.Images removed due to copyright reasons.Hamster pancreatic NucleusMitochondrionEarliest Time pointNextobservedlocationLocationAfterGolgiMillstein“in vitro synthesis of immunoglobulin light chains. …To our delight we ran into the unexpected observationof the existence of a biosynthetic precursor oflight chains. Further experiments led us to proposethe extra N-terminal sequence was a signal forvectorial transport across the membrane duringprotein synthesis. That was the first evidence whichindicated that the signal for secretion was an N-terminalsegment, rapidly cleaved during protein synthesis.”FROM NOBEL LECTURE 1984Image removed due to copyright reasons.CytoplasmicExtractsNMessenger RNARibosomes &charged tRNAsMicrosomes(RER vesicles)in vitroBlobelImage removed due to copyright reasons.+added late+++added early--Purifiedextract++-Microsomes+++MessageRibosomestRNAsProtein in supernatentProtein in lumen ofmicrosomesProtein insupernatentNProtein in supernatentFrom the previous experiment, Blobel demonstrated that the amino acidsequence at the beginning (N terminus) of exported proteins is recognizedby a complex.This complex is required to get the protein into the lumen of ER.To get into the lumen of the ER the protein has to be just beginning to betranslated.Since not all exported proteins have the same N terminus, Blobelpredicted, like Millstein, whatever the sequence was, it would be latercleaved.Gunter BlobelNobel Laureate, 1999Image removed due to copyright reasons.+Hydrophobic amino acidsN~20 amino acids5’“Signal Sequence”Bacterium Eukaryotic CellNNNSRPSignalSequenceSignal RecognitionParticleSRP receptor“DockingProtein”SRP receptor“DockingProtein”TransloconSRP receptor“DockingProtein”SRP receptor“DockingProtein”Signal peptidasecleaves off the signalSRP receptor“DockingProtein”Fully secreted ProteinSRP receptor“DockingProtein”Signal peptidasecleaves off the signalSRP receptor“DockingProtein”Membrane proteinEUKARYOTIC CELLPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellTransportVesiclesEUKARYOTIC CELLPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellEUKARYOTIC CELLPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellEUKARYOTIC CELLPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellSecretoryVesiclesPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellSecretoryVesiclesPlasmamembraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellPlasma membraneEndoplasmicRecticulumGolgiApparatusCytoplasmOutside the CellTransportvesiclesSecretoryVesiclescytoplasmchromosomePeriplasmInner membraneOuter membrane-galactosidaselacZHow were the Secgenes identified?BacteriummRNACytoplasmchromosomeInner membrane-galactosidaselacZHow were the Secgenes identified?Active -galactosidase is a tetramer.This cell can utilize lactose as a carbon source.LAC+Gene encoding Exported Proteinlac Z gene5’3’5’ 3’5’ 3’Gene FusionThe 5’ end of the coding region oflac Z is fused tothe 5’ end of a gene encoding an exported protein including the signal sequence.‘lac Z gene5’ 3’Gene FusionWhere the 5’ end of thelac Zgene is fused to the 5’ end of agene encoding an exported protein including the signal sequence.This gene fusion results in a hybrid protein where theN-terminus of -Galactosidase is fused with a signal sequence.Signal SequenceNC-galactosidaseCytoplasmchromosomePeriplasmInner membraneOuter membraneHybrid proteinGene fusionThe hybrid protein protein localizes to the membrane.This cell is unable to utilize Lactose as a Carbon Source.LAC-Cells with this gene fusion are…CytoplasmchromosomePeriplasmInner membraneOuter membraneGene fusionLAC- LAC+>95% of the Lac+ mutants have mutations …..linked to the gene fusion resulting in ….?XHybrid proteinXXXXJon Beckwith+Hydrophobic amino acidsN~20 amino acidsCXCytoplasmchromosomePeriplasmInner membraneOuter membraneGene fusionHybrid proteinLAC- LAC+XXXXCytoplasmchromosomePeriplasmInner membraneOuter membraneHybrid proteinGene fusionLAC- LAC+XXXXConditional LethalHow to get get Mutations in essential genesTemperature- sensitiveCold-sensitive20°C 37°CActive InactiveActiveConditional LethalHow to get get Mutations in essential genesTemperature- sensitiveCold-sensitive20°C 37°CActive InactiveInactiveActiveSec ASec BSec DSec ESec GSec YPost-TranslationalPost-translationalCotranslationalTranslationalstate ofprotein inchannelWhat is theEnergy Source?How does theprotein crossthe membrane?SignalDestinationGTP hydrolysisATP hydrolysisPowered bytranslationImportinsdeliver toNuclear PoreComplex (NPC)Chaperones bindProtein entersMito. ChannelSRP binds SSSRP binds DPProtein enterschannelNuclearLocalizationSignal (NLS)7aa + chargedN-terminalAmphipathicHelix20-50 aaCotranslationalPowered bytranslationSRP binds SSSRP binds DPProtein enterschannelSignal SequenceSignal SequenceNucleusMitochondrionOutside thecellPlasmamembraneNOYesYesYesSignal
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