1. Post-Golgi biosynthetic pathways

2. MDCK-cell Resting fibroblast Migrating fibrobl. The epitelial cell line MDCK is the most studied model system for polarised sorting and transport.

3. Hepatocyte Retinal pigment Retinal rod cell epitelial cell

4. Hippocampus neuron OsteoclastBudding yeast cell

5. Sorting along the biosynthetic pathway in epitelial cells. MDCK-cells as model system. Sorting in the trans- Golgi network. THE SORTING DEPENDS ON SIGNALS IN THE MOLECULES TO BE SORTED

6. Classical signals: *Sorting of lysosomal enzymes to lysosomes *Basolateral transport in epithelial cells. *Retrograde transport from the Golgi to ER. *Endocytosis of receptors and other molecules from the cell surface – a fraction is sorted to the trans-Golgi network.

7. Before any basolateral sorting signals were identified (1991 ->), it was suggested that basolateral transport occured by “bulk flow” while transport to the apicale side – which is the specialised domain in epithelial cells – would require sorting. In 1991 it was published, however, that the transmembrane protein pIgA receptor was transported basolaterally in a signal dependent manner.

8. Growth of MDCK epitelial cells on filters. Transfer to glass-dishes with 90 ml of medium for establishment of confluent cell layers.

9. Basolateralt medium Apikalt medium

10. PROTEIN SORTING STUDIES IN THE SECRETORY PATHWAY OF EPITHELIAL CELLS A protein transported equally well to the apical or basolateral surfaces is regarded as a protein without a sorting signal. Secretory proteins are the ones most easily studied, because these may be identified after harvest of the two opposite media of filter-grown cells. Apical or basolateral membrane proteins may be extracted after differetial biotinylation of the two opposite sides and/or recognition by specific antibodies. Sorting signals are autonomous – they work after ”transplantation” to normally non-sorted molecules.

11. Baso Api Baso Api Baso Api Lumenal domain tm Cytoplasmic domain

12. Golgi membrane /endosomal membrane Cytoplasm Lumen Cytoplasmic domain Transmembrane domain Lumenal domain

13. Golgi membrane /endosomal membrane Cytoplasmic domain TM domain Lumenal domain Truncated variant Example: Polymeric IgA receptor = pIgR Basolateral Apical

14. Golgi membrane /endosomal membrane Cytoplasmic domain TM domain Lumenal domain Truncated variant Example: Polymeric IgA receptor = pIgR Basolateral Apical 3 aa

15. Golgi membrane /endosomal membrane Cytoplasmic domain TM domain Lumenal domain Example: Polymeric IgA receptor = pIgR Basolateral Apical Recessive apical sorting information

16. Golgi membrane /endosomal membrane Cytoplasmic domain TM domain Lumenal domain Deletion of amino acids 655-668 = 14 amino acids Basolateral Apical 17 aa

17. Golgi membrane /endosomal membrane Cytoplasmic domain TM domain Lumenal domain 17 amino acids: RARHRRNVDRVSIGSYR, red identified by ala scan Basolateral Baso Apical

18. Golgi membrane /endosomal membrane TM domain Lumenal domain Apical Baso Apical Reporter protein: PLAP (placental alkaline phosphatase) extracellular (lumenal) domain

19. WHAT MOTIFS ARE BASOLATERAL SORTING SIGNALS? YXX  (YVEL/YTDI/YXRF)  bulky/hydrophobic TGN38, M6PR LL / IL / LEL Fc Receptor, MHC II Invariant chain, EGF receptor NPXY Low density lipoprotein (LDL) receptor H/R-XXV Polymeric IgA receptor, Mannose-6-phosphate rec. (?) PXXP Epidermal growth factor (EGF) receptor Other Transferrin receptor (VDGDNSHVEMKLA) Some proteins have several sorting signals in their cytoplasmic tail. These are recognized at different intracellular sites, to provide recycling to the basolateral domain.

20. BASOLATERAL SORTING SIGNALS Some basolateral sorting signals overlap with endocytosis signals. *Fc receptor *Asialoglycoprotein receptor *Lysosomal acid phosphatase Other basolateral sorting signals are distinct from endocytosis signals. *Polymeric IgA receptor *LDL receptor *Transferrin recptor (?)

21. HOW AND WHERE IS BASOLATERAL SORTING INFORMATION RECOGNIZED? Adaptor complexes 1-3 Lysosomal route Basolateral route via endosomes

22. The first proteins regarded as mediators of basolateral sorting were adaptins – already known to be involved in endocytosis from clathrin coated pits at the cell surface. The adaptins consist of 2 large, 1 medium and 2 small subunits. 4 different adaptin- complexes have been discovered. AP-1A: TGN (+ endosomes) AP-1B: Epithelia specific AP-3A: Endosome/TGN AP-3B: Neuron-specific (endosome?) A/B Baso

23. AP-1, AP-2 and maybe AP-3 (in mammals) may bind clathrin. All 4 complexes are found in Arabidopsis, but only AP 1-3 in Drosophila. Many of the subunits are found as closely related isoformes coded by separate genes making a large number of combinations possible. Endocytosis

24. AP-1B contains a specific  1B subunit which only is expressed in certain polarised cells (not all polarised cell types, mainly epithelia). Recognizes tyr-based signals. AP-4 has also been connected to basolateral sorting, but has equal or overlapping specificity with AP-1B. There is still room for more adaptors for basolateral sorting. AP-4 Somewhere in the picture: FAPP1 and FAPP2, mediating TGN => PM transport. FAPP2

25. GGA (1-3): Golgi-associated,  -adaptin homologous, ARF-interacting proteins N-terminal hydrofobic sequence ARF-1 GDP GEF AP-1 What about tyr-signals? Ubq Rabaptin 5 binding Ear = GAE  -synergin?

26. Dynamin Cortactin Actin Membrane Arp 2/3 complex

27. At least 6 ARFs exist in mammals, 5 are localised to the Golgi-apparatus and 1 to the plasma membrane. Aktiv membranbundet cytoplasmatisk 4 families GEFs with several members ARF1-GTP (myristoylated), a tyrosine based signal, and phosfatidylinositol 4,5 bisphosphate are necessary to recruit AP-1 clathrin adaptors to membranes. Phosphatidylinositides of the 4-series has been regarded as important for Golgi.

28. GGA dependent receptors CI-Mannose-6-phosphate receptor CD-Mannose-6-phosphate receptor Sortilin SorLA/LR11 LRP-3  -secretase

29. Clathrin- dependent Non-clathrin dependent Some transmembrane proteins have cytoplasmic domains that interact directly with microtubule motors

30. What about APICAL SORTING IN EPITHELIAL CELLS? Glycans: N-glycans, O-glycans, CS glycosaminoglycans Yes (maybe and no), yes (maybe), yes. GPI-anchors? NOT REALLY – OR? LIPID DOMAINS?? Protein motifs for apical sorting: Megalin NPXY. The second of three NPXY motifs is crucial (distance from membrane…).

31. MDCK cells transfected with the gene for the non- glycosylated protein rat growth hormone (rGH) secretes this protein randomly, which is slightly more basolaterally rGH with 2 N-linked glycosylation sites is secreted almost exclusively into the apical medium.

32. Erythropoietin – three N-glycans, one is critical. Endolyn – eight N-glycans, not all equally important. O-glycans of mucin type may also mediate apical sorting: Intestinal sucrase-isomaltase Gp-40 Several other examples But several examples of non-sorted glycoproteins also exist.

33. HS CS

34. Transmembrane lectin-molecule raft VIP 36? A Hypothesis Detergent insoluble proteins of apical transport vesicles were separated by 2D-gel analysis and sequenced. One putative lectin molecule was found: VIP 36.

35. WHAT IS A (GLYCOLIPID) RAFT? Glycolipid- and cholesterol rich domains in a lipid membrane are associated in a more stable structure than lipids are according to “the fluid mosaic model.” On the cell surface of a “regular” cell, these domains will have a diameter of 60 - 100 nm. In specialised membranes may larger areas of the plasma membrane have raft-characteristics. Example: The apical membrane of epithelial cells (MDCK). Do lipids and lipid-binding proteins play a role in sorting of molecules that are transported from the TGN to the apical membrane?

36. Caveolins : Proteins with affinity for specialised lipid- domains. Palmitoylation. Might be necessary for cell surface transport of GPI-anchored proteins. Not apical transport. GPI-anker : Glycosyl-phosphatidyl-inositol-anchor that might bind proteins to a membrane. For some time regarded as sorting signals for apical transport, since these proteins usually are localised to rafts. The apical sorting is most likely dependent on N-glycans (via transcytosis?). Glycosphingolipids: are glycolipids that are mainly transported to the apical side in MDCK-cells (from the TGN). Present in rafts rich in cholesterol. MAL (VIP 17): A protein that seems to mediate apical sorting of several cargo proteins in MDCK-cells.

37. There are probably several independent transport mechanisms operating in parallell, both to the apical and to the basolateral side of MDCK epitelceller. The apical ones may be raft-based or not raft-based.

38. Annexin II, Annexin XIIIb, FAPP1/2, MAL (VIP17) Protein kinase D

39. All mechanisms for sorting from the TGN are not known *We have only discussed proteins with one transmembrane domain, while many proteins span the membrane several times. These may also be sorted. How? *Some apical proteins, like megalin, have been reported to have signals in the cytoplasmic tail (interaction with motor proteins?) *Ubiquitinylation may shift the sorting from TGN to the plasma membrane towards TGN to lysosomes (the vacuole in yeast). *Lipids may play a role in sorting in many ways. *What factors are necessary for budding, transport and fusion? (v- and t- SNARES, GTP-binding proteins, etc.)