Slide 1Slide 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 35Modified tRNAs allowed isolation (cross-linking approach)and analysis (fluorescence quenching ) of translocation channelIn 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 ratchetting (posttranslational)From: Beckmann et al. Cell (2001) Vol 107, 361-372From: Beckmann et al. Cell (2001) Vol 107, 361-372Topology of membrane-spanning proteins-> Topogenic sequences determine orientationType I membrane proteins have a cleavable signal sequenceStop transfer sequence (hydrophobic): when mutated -> secretionType II membrane proteins have internal signal sequenceInternal signal is not cleaved. Direction is determined by residues outsideof hydrobhobic helix (++-outside rule: postive aa face cytoplasm)From: Beckmann et al. Cell (2001) Vol 107, 361-372Translocation of proteins with multiple membrane spanning domainsFormation 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/ThrMoviesQuickTime™ 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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