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Published byΠαρθενιά Δυοβουνιώτης Modified over 6 years ago
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Modified tRNAs allowed isolation (cross-linking approach)
and analysis (fluorescence quenching ) of translocation channel
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In vitro reconstitution of ER translocation:
- 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 ratchetting (posttranslational)
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From: Beckmann et al. Cell (2001) Vol 107, 361-372
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From: Beckmann et al. Cell (2001) Vol 107, 361-372
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Topology of membrane-spanning proteins
-> Topogenic sequences determine orientation
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Type I membrane proteins have a cleavable signal sequence
Stop transfer sequence (hydrophobic): when mutated -> secretion
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Type II membrane proteins have internal signal sequence
Internal signal is not cleaved. Direction is determined by residues outside of hydrobhobic helix (++-outside rule: postive aa face cytoplasm)
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From: Beckmann et al. Cell (2001) Vol 107, 361-372
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Translocation of proteins with multiple membrane
spanning domains
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Formation of a glycosylphosphatidylinositol (GPI)-anchor
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ER 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!)
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Folding of Influenza hemagglutinin (HA)
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Ser/Thr
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Movies
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Summary ER translocation: SRP-dependent and -independent pathways; translocation occurs through Sec61 complex; topogenic sequences determine overall orientation. ER function Compartmental identity: maturation versus fixed compartments Identification of components: combination of genetics, biochemistry... Vesicular coats: COPI ~ retrograde: Golgi->ER COPII ~anterograde: ER->Golgi CCV post-Golgi, various adaptors
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