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

  2. The major trafficking pathways in polarized epithelial cells.

  3. Mammals Yeast Localisationof differentphosphoinositides, kinasesandphosphatases. If lipid domains are formed withparticular phosphoinositidesearly in the secretory pathway?

  4. Phosphoinositidesare precursors (forløpere) for 2nd messengers and attachment sites for proteins Phosphorylationofphosphatidyl-inositol (PI) givesphosphoinositidesofdifferent kinds. Viktig for transport fra TGN til vakuolen Kinasesthatphosphorylate PI have been found in ER, the nucleus, Golgi, endosomesand the plasma-membrane. The kinases have different specificities – producing different products. LPI: Lysophosphatidylinositol GPI: Glycerophosphatidylinos (Proteinanchorsformed in ER)

  5. ARFs can bind to phosphoinositides alone or in complex with other proteins PI(4,5)P2 indifferent vesicular transport processes. Thelipid forms sites for membrane pinching or fusion. These sites might be localised to raft-like domains. ARF-GTPasesare necessary for recruiting many proteins – also PIP kinases.

  6. Prydz and Simons. 2001. Biochem. J. A B A B

  7. Do stabilised lipid domains form early in the Golgi apparatus? Emery G, Parton RG, Rojo M, Gruenberg J. The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics.J Cell Sci. 2003 Dec 1;116(Pt 23):4821-32.

  8. *What makes a Golgi-enzymeremain in the Golgiapparatus (longer than proteins in transit)? *What keeps the Golgi cisternaetogether? A typical Golgi glycosyl-transferasespans the membrane once and has the N-terminalend of a short cytoplasmic tail (class II protein) There are probably hundreds of glycosyltransfarases in a mammalian Golgi apparatus.

  9. Sean Munro observed that proteins in the plasmamembrane have a longer hydrofobic transmembrane domain than the Golgienzymes. Impact on localization?

  10. Munro also observedan overrepresentation ofa tyr or trp residueafter the hydrofobic sequence. Impact on localization?

  11. Medial Golgi enzymesfor higher order complexes: Exampel: N-acetylglucosaminyltransferase I (NAGT I) and mannosidase II (Mann II). Trans-Golgi enzymesdo not form such complexes. The luminal domainsare important forcomplex formation, but is the transmembrane domain also important?

  12. Enzymeslate in the Golgi-apparatus havebeen relativelyeasyto study: Example: 2,6-sialyltransferase localizes to the trans-regionof the Golgi by means of the 17-aa transmembrane domain. Enzymesearlier in the Golgi-apparatusthat have been expressed indifferent celllinesdid often seem 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 constitutethe glycosyltransferase activitywhen heparansulfate is polymerised. Must form hetero-oligomeric complexes to leave the ER.

  13. OPEN QUESTIONS: What is the fraction of Golgienzymes passingtheirproperlocalisation to be retrieved by COP I containing vesicles? Or if not retrieved in COP I vesicles, what is the mechanism? Do retrogradely transported vesicles pass several cisterneato fuse with an earlier compartment – only to allow enzymes to move anterogradely towards their proper localisation? If the vesicles do not contain Golgi enzymes, what do they contain?

  14. Localisationof 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 plasmamembrane. 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 geneshave been identified in yeast (Saccharomyces cerevisiae) wheremutations make the cells incapable of retaining proteins localised late in the Golgiapparatus.

  15. While enzymes in the Golgi apparatus return to the ER upontreatment with Brefeldin A, does one class of proteins remain:Golgi-MATRIX-proteins.It seems that these proteins govern the appearance and function of the Golgi. ER exit sites, ERGIC and 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.

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

  17. Not mentioned (so far): P24/P25 proteins – a group of proteinswith Mw of 23 - 27 kDa that form heteromere complexes. These proteins are involved in COP II dependent transport from the ER to the Golgi and COP I dependent retrograde transport to the ER. Deletion of KKXX motif in P25 (=> SSXX) results in apical localisation of both P25 and P24. Regulation of cholesterol content in Golgi membranes?