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Modified tRNAs allowed isolation (cross-linking approach) and analysis (fluorescence quenching ) of translocation channel.

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Presentation on theme: "Modified tRNAs allowed isolation (cross-linking approach) and analysis (fluorescence quenching ) of translocation channel."— Presentation transcript:

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3 Modified tRNAs allowed isolation (cross-linking approach)
and analysis (fluorescence quenching ) of translocation channel

4 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)

5 From: Beckmann et al. Cell (2001) Vol 107, 361-372

6 From: Beckmann et al. Cell (2001) Vol 107, 361-372

7 Topology of membrane-spanning proteins
-> Topogenic sequences determine orientation

8 Type I membrane proteins have a cleavable signal sequence
Stop transfer sequence (hydrophobic): when mutated -> secretion

9 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)

10 From: Beckmann et al. Cell (2001) Vol 107, 361-372

11 Translocation of proteins with multiple membrane
spanning domains

12 Formation of a glycosylphosphatidylinositol (GPI)-anchor

13 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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15 Folding of Influenza hemagglutinin (HA)

16 Ser/Thr

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24 Movies

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35 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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