1How many proteins are there in a cell?# of genes in genome?25-30,000Amount of protein/cell?~0.8 ngLet’s assume 10,000 expressed in a given cellThat converts to 1010 molecules of 50kDa per cellIf all were equally abundant - 106 (1010/104)of each per cellOf course, they vary widely in size and abundance2D Gel Analysis2Abundant proteins are easy to purify -Actin is 10% of many cells - 10x purification -> purityBUT - most proteins are not as abundantA major cell surface receptor - ~106 molecules per cellor 10-4 of total cellular proteinSo need 104 x purificationAssume 10% yieldSo 1g of cell protein (109 cells = 100 x 100mm dishes)will yield 1g x 10-4 x 0.1 = 10ug1pmol = 50ng of a 50kDa proteinBut what if it were 100-1000 x less abundant?3A minor cell surface receptor ~100-1000 copies per cell -need 107-108 fold purification.Animal tissue often a better source than cellsAlternative approaches:-affinity purificationmass spectrometryclone the gene firstexpression or functional cloninggenomicsthen express the proteinbacteria, yeastanimal cellsanimals (transgenesis)Purification of Membrane Proteins: Purification of Membrane Proteins: immunoaffinity immunoaffinity chromatographychromatographyPeptide-ElutedFractionsCellLysateFlow ThroughWash20011697664531kDamSR-BI-t1123456784Analyses of protein functionBiochemistry - purification and assayExpression - transformation, transgenesisInhibition - genetics (forward and reverse) - drugs, antibodies, - “dominant negatives” - RNAi, antisenseMembranesPlay key roles in cell functioncompartmentalizationtransportintercellular signallingcell adhesionMembrane proteins have special properties bothbecause of the membrane environment andin line with their different functions5RepresentativeRepresentativeIntegralIntegralMembraneMembraneProteinsProteins6Two subunits of the Two subunits of the KcsA KcsA KK++ channel channelSpecificity Filter (orange)Helical Gatehttp://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v417/n6888/full/417523a_fs.html&filetype=&_UserReference=C0A804ED4654D463FE606837EB983D756FD8Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)7*LnRnGnYCHANNELSAC cAMPcAKinasePDE cGMPcGKinaseCHANNELSPLAPLCArachidonicacidProstaglandinsEicosanoidsPIP2IP3Ca++CaMKinaseDAGKinase CPI derivativesKinase B/AktPI3K8Membrane Trafficking andMembrane Trafficking andthethe Secretory Secretory PathwayPathway::ER->ER->GolgiGolgi->out->outSorting to differentSorting to differentmembrane-boundedmembrane-boundedorganellesorganellesHow is this all orchestrated?How is this all orchestrated?How do we know?How do we know?9Image of outer surface of theImage of outer surface of thecell by cell by Heuser Heuser method:method:Low Density LipoproteinLow Density Lipoprotein(LDL, 20 nm) bound to surface(LDL, 20 nm) bound to surfacereceptors clustered in receptors clustered in ‘‘CoatedCoatedPitsPits’’LDL100 nmImage of inner surface of theImage of inner surface of thecell by cell by Heuser Heuser method:method:Clathrin Clathrin Coated PitCoated Pit100 nm10Reconstructed Image ofReconstructed Image of clathrin clathrin shell from EMshell from EM21 A resolution21 A resolution11Blood cells circulate around thebody and stop at appropriate placesHOW DO THEY KNOW WHERE TO GO?LEUKOCYTEADHESIONPLATELETADHESIONROLLINGADHESIONEXTRAVASATIONINVASIONCELL ADHESION in theVASCULAR SYSTEMIntegrin extracellular heterodimerIntegrin extracellular heterodimer12Intravital Microscopy ofLeukocytes in VenulesSome cells move about the bodyHOW DO THEY ATTACH and DETACH and HOW DO THEY MOVE?SEPARATION ofGROUPS of CELLSMIGRATIONS ofINDIVIDUAL CELLSPROJECTION ofNERVE PROCESSES13Lamellipodial Lamellipodial ProtrusionProtrusionedge ofedge oflamellipodiumlamellipodiumfocal adhesionsfocal adhesionsMenaActinFrank GertlerHippocampalHippocampalGrowth ConeGrowth ConeGeraldine StrasserGeraldine Strasser14Actin Assembly Drives Lamellipodial ProtrusionBarbed EndsFrank GertlerBrain Development Requires Cell Migration and Axon GuidanceFrank Gertler15NeuronsCell migration to reach appropriate placesAxonal projections to appropriate targetsSynaptic communicationOrganization of pre- and postsynapticmembranesNext week - membrane structureHow do we know that membranes are lipid bilayers?How does that impact membrane protein structure/function?Protein-lipid and protein-protein
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