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!)

15. Folding of Influenza hemagglutinin (HA)

16. Ser/Thr

24. Movies

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