1. RNA localization mRNA can be localized to subcellular compartments by actin or tubulin-dependent processes Examples: Xenopus: Vg1 mRNA (TGF  ) to vegetal pole Drosophila: nanos, oskar mRNA (posterior) and bicoid (anterior) (requires mRNA binding protein staufen) (requires staufen and miranda) prospero (into ganglion of mother cells; neuroblast TF) Yeast: Ash1 mRNA to daughter cell

3. lamellipodia stainingperinuclear staining in myotubes

4. Bertrand et al., Mol Cell (98) 2:437-445

6. SUMMARY 2 I.mRNA decay - regulated and non-regulated turn-over - ordered pathway (deadenylation, decapping, exonucleolytic degradation) - NMD: recognition of premature stop codons II.Cytoplasmic mRNA localization - ZIP code in 3’ UTR - both actin and tubulin-mediated - yeast mating type switch as a model: Ash1 mRNA localization (via 3’ UTR, She2/3, Myo4 and actin cables)

7. ER translocation & vesicular transport

10. from: Jamieson and Palade 3min 7min 37min117min

20. In 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 ratcheting (posttranslational)

21. Probing of translocation intermediates with fluorescent peptides From: Liao and Johnson Cell (97)

22. The Sec61 complex forms a channel Menetret et al. Mol Cell (2000) 6:1219

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

25. From: Van den Berg et al. Nature (2004) 427, 36-44

27. Topology of membrane-spanning proteins

28. Type I membrane proteins have a cleavable signal sequence

29. Type II membrane proteins have internal signal sequence

30. Type III membrane proteins have internal signal sequence

31. Type II+III membrane proteins have internal signal sequences

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

33. Translocation of proteins with multiple membrane spanning domains

34. From: Van den Berg et al. Nature (2004) 427, 36-44

35. Formation of a glycosylphosphatidylinositol (GPI)-anchor

36. 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!)

38. Folding of Influenza hemagglutinin (HA)

39. Ser/Thr

57. 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