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.

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

6. Phosphoinositides are precursors (forløpere) for
2nd messengers and attachment sites for proteins
Phosphorylation of phosphatidyl-inositol (PI) gives phosphoinositides of different kinds.
Viktig for transport fra TGN til vakuolen
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.
LPI: Lysophosphatidylinositol
GPI: Glycerophosphatidylinos
(Protein anchors formed in ER)

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

9. Prydz and Simons. 2001. Biochem. J.

10. 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 Dec 1;116(Pt 23):
Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K.
Related Articles, Links Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K.
   
Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci U S A Mar 28;97(7):

11. *What keeps the Golgi cisternae together?
*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 probably hundreds of glycosyltransfarases in a mammalian Golgi apparatus.

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

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

14. 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?

15. 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 did 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 constitute the glycosyltransferase activity when heparan sulfate is polymerised. Must form hetero-oligomeric complexes to leave the ER.

17. OPEN QUESTIONS:
What is the fraction of Golgi enzymes passing their proper localisation 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 cisternea to 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?

18. 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.

20. While enzymes in the Golgi apparatus return to the ER upon treatment 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 GM p rab 1
Medial-Golgi: GRASP Golgin rab 2
The Golgi-apparatus collapses without Golgin-45. Golgin-45 does not return to the ER upon BFA-treatment.

21. 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.

23. Not mentioned (so far):
P24/P25 proteins – a group of proteins with Mw of 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?