PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57RNA localizationmRNA can be localized to subcellular compartmentsby actin or tubulin-dependent processesExamples:Xenopus: Vg1 mRNA (TGF) to vegetal poleDrosophila: nanos, oskar mRNA (posterior) and bicoid (anterior)(requires mRNA binding protein staufen)(requires staufen and miranda) prospero (into ganglion of mother cells; neuroblast TF) Yeast: Ash1 mRNA to daughter celllamellipodia staining perinuclear staining in myotubesBertrand et al., Mol Cell (98) 2:437-445SUMMARY 2I. mRNA decay- regulated and non-regulated turn-over- ordered pathway (deadenylation, decapping, exonucleolytic degradation)- NMD: recognition of premature stop codonsII. Cytoplasmic mRNA localization- ZIP code in 3’ UTR- both actin and tubulin-mediated - yeast mating type switch as a model: Ash1 mRNA localization (via 3’ UTR, She2/3, Myo4 and actin cables)ER translocation & vesicular transportfrom: Jamieson and Palade3min7min37min 117minNNNONNH2HNNNNHOOHN Ala 151HN Ala 151OOCAsp 125Val 124Lys 123Asn 122NH3+OH2NCAsn 125Val 124Lys 123Asn 122NH3+Ran WTRan WTRan D125NRan D125NGTPGTPXTPXTPIn vitro reconstitution of ER translocation:- Sec61 complex: conserved translocation channel Sec61 subunits () Sec62/63 TRAM (translocating chain-assoc. membrane protein)- phospholipids (proteoliposomes) and luminal chaperones (BIP)- SRP/SRP receptor only required for co-translational translocation not for post-translational translocation (e.g pre-pro-alpha factor).- energetics of translocation: protein conducting channel (cotranslational) molecular ratcheting (posttranslational)Probing of translocation intermediates with fluorescent peptidesFrom: Liao and Johnson Cell (97)The Sec61 complex forms a channelMenetret et al. Mol Cell (2000) 6:1219From: Beckmann et al. Cell (2001) Vol 107, 361-372From: Beckmann et al. Cell (2001) Vol 107, 361-372From: Van den Berg et al. Nature (2004) 427, 36-44From: Van den Berg et al. Nature (2004) 427, 36-44Topology of membrane-spanning proteinsType I membrane proteins have a cleavable signal sequenceType II membrane proteins have internal signal sequenceType III membrane proteins have internal signal sequenceType II+III membrane proteins have internal signal sequencesFrom: Beckmann et al. Cell (2001) Vol 107, 361-372Translocation of proteins with multiple membrane spanning domainsFrom: Van den Berg et al. Nature (2004) 427, 36-44Formation of a glycosylphosphatidylinositol (GPI)-anchorER function- Proper folding of proteins (chaperones, lectins, petidyl-prolyl-isomerases)- Formation of disulfide bonds (PDI) GSH prevents oxidation in cytosol GS-SG + NADPH + H+ <=> 2 GSH + NADP+- Proteolytic cleavages- Addition & processing of carbohydrates- Assembly into multimeric proteins- Ca2+ storage- Lipid synthesis- Detoxification (liver!)Folding of Influenza hemagglutinin (HA)Ser/ThrQuickTime™ and aGIF decompressorare needed to see this picture.QuickTime™ and aGIF decompressorare needed to see this picture.QuickTime™ and aGIF decompressorare needed to see this picture.SummaryER translocation: SRP-dependent and -independent pathways; translocation occurs through Sec61 complex; topogenic sequences determine overall orientation.ER functionCompartmental identity: maturation versus fixed compartmentsIdentification of components: combination of genetics, biochemistry...Vesicular coats: COPI ~ retrograde: Golgi->ER COPII ~anterograde: ER->Golgi CCV post-Golgi, various
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