1. TGN SNAP-23, syntaxin-3 (t) VAMP-7 (v) VAMP-8 (?) Annexin 13B MAL 17 Protein kinase D NSF,  -SNAP, syntaxin 4(?), SNAP 23, VAMP (toxin sensitive), cdc42 (Rho GTPase), lin genes, exocyst, calmodulin ? m.m. Annexin II

2. The major trafficking pathways in polarized epithelial cells.

3. Yeast Mammals Localisation of different phosphoinositides, kinases and phosphatases. If lipid domains are formed with particular phosphoinositides early in the secretory pathway?

4. Phosphoinositides are precursors (forløpere) for 2nd messengers and attachment sites for proteins Phosphorylation of phosphatidyl- inositol (PI) gives phosphoinositides of different kinds. Kinases that phosphorylate PI have been found in ER, the nucleus, Golgi, endosomes and the plasma-membrane. The kinases have different specificities – producing different products. (Protein anchors formed in ER) Viktig for transport fra TGN til vakuolen LPI: Lysophosphatidylinositol GPI: Glycerophosphatidylinos

6. *What makes a Golgi-enzyme remain in the Golgi apparatus (longer than proteins in transit)? *What keeps the Golgi cisternae together? A typical Golgi glycosyl- transferase spans the membrane once and has the N-terminal end of a short cytoplasmic tail (class II protein) There are hundreds of glycosyltransferases in a mammalian Golgi apparatus.

7. Sean Munro observed that proteins in the plasma membrane have a longer hydrofobic transmembrane domain than the Golgi enzymes. Impact on localization?

8. Munro also observed an overrepresentation of a tyr or trp residue after the hydrofobic sequence. Impact on localization?

9. Medial Golgi enzymes for higher order complexes: Exampel: N-acetylglucosaminyltransferase I (NAGT I) and mannosidase II (Mann II). Trans-Golgi enzymes do not form such complexes. The luminal domains are important for complex formation, but is the transmembrane domain also important?

10. Enzymes late in the Golgi-apparatus have been relatively easy to study: Example:  2,6-sialyltransferase localizes to the trans-region of the Golgi by means of the 17-aa transmembrane domain. Enzymes earlier in the Golgi-apparatus that have been expressed in different cell lines often seemed to localize to the ER. Eventually it became evident that these enzymes needed partner-enzymes to be able to leave the ER in hetero- oligomeric complexes. Example : EXT 1 and EXT 2: Two proteiner that together constitute the glycosyltransferase activity when heparan sulfate is polymerised. Must form hetero-oligomeric complexes to leave the ER.

12. OPEN QUESTIONS: What is the fraction of Golgi enzymes passing their proper localisation before retrieval by vesicles or tubules? Do retrogradely transported vesicles or tubules bypass several cisternea to fuse with an earlier compartment – only to allow enzymes to move anterogradely towards their proper localisation? Do stable lipid domains form early in the Golgi apparatus (or already in the ER), allowing segregation of apical and basolateral pathways to take place before the trans-Golgi network?

13. Localisation of proteins to the trans-Golgi network: This compartment is the “exit site” from where transport in several different directions occur. It is probably not avoidable that enzymes acting in this compartment to a large extent are transported to the plasma membrane. Naturally, endocytosis signals and signals for transport to the TGN have been found in such enzymes (example, the protease furin). Retention of proteins late in the Golgi may be quite complicated. 18 different genes have been identified in yeast (Saccharomyces cerevisiae) where mutations make the cells incapable of retaining proteins localised late in the Golgi apparatus.

15. While enzymes in the Golgi apparatus return to the ER upon treatment with Brefeldin A, one class of proteins remains in position: Golgi-MATRIX-proteins. It seems that these proteins govern the appearance and function of the Golgi. Cis-Golgi: GRASP 65 + GM 130 + p 115 + rab 1 Medial-Golgi: GRASP 55 + Golgin-45 + rab 2 The Golgi-apparatus collapses without Golgin-45. Golgin- 45 does not return to the ER upon BFA-treatment. ER exit sites, ERGIC and Golgi

16. Golgi-cisternae are so close together that it seems as if vesicle budding and fusion only might be feasible at the ends of the cisternea.

18. Golgi apparatus VTCs COP II COP I Endoplasmic reticulum

19. COP II vesicle p115 Rab 1 COP II vesicle +GTP