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 32The Secretory Pathway- Classic Experiments- ER Translocation- Membrane budding and fusionIntracellular Transport PathwaysIntracellular Transport PathwaysVesicular TransportVesicular TransportPulse-chase: Palade and co-workers3H-Leu pulse to monitor secretion in pancreatic cells10’ Golgi45’ Secretory GranulesModern version: GFP allows visualization of transport1. ts VSV G accumulates in ER2. After lowering T: ER exit sites3. Golgi4. Plasma membraneGenetics: Schekman and co-workersNovick et al. 1980: Identification of 23 SEC genesGenetics: Schekman and co-workersIn vitro assay for vesicular transport: Rothman and co-workersBalch et al. 1984In vitro assay for ER translocation: Blobel and co-workersIn vitro assay for ER translocation: Blobel and co-workersIdentification of signal sequencesSRP: signal recognition particleSRP cycleSRP cycleSite-specific incorporation of modified amino acids to probe ER translocationER translocation can be reconstituted in vitro:- Sec61 complex: conserved translocation channel Sec61 subunits (a, b, g) 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 channel….Menetret et al. Mol Cell (2000) 6:1219From: Beckmann et al. Cell (2001) Vol 107, 361-372The translocon by EM tomographyFrom: Beckmann et al. Cell (2001) Vol 107, 361-372The translocon by EM tomographyFrom: Van den Berg et al. Nature (2004) 427, 36-44X-ray structure of the prokaryotic translocation channel SecYFrom: Van den Berg et al. Nature (2004) 427, 36-44Model for translocation of soluble proteinsTopology 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, 361-372Model for translocation of transmembrane proteinsTranslocation of proteins with multiple membrane spanning
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