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Post-Golgi biosynthetic pathways

Post-Golgi biosynthetic pathways. MDCK-cell Resting fibroblast Migr ating fibrobl. The e pitel ial cell line MDCK is the most stud ied model system for polaris ed sort ing and transport. Hepatocyt e Retina l pigment Retina l rod cell

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Post-Golgi biosynthetic pathways

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  1. Post-Golgi biosynthetic pathways

  2. MDCK-cell Resting fibroblast Migrating fibrobl. The epitelial cell line MDCK is the most studied modelsystem for polarised sortingand transport.

  3. Hepatocyte Retinal pigment Retinalrod cell epitelial cell

  4. Hippocampus neuron Osteoclast Budding yeast cell

  5. Sortingalong the biosynthetic pathway in epitelial cells. MDCK-cellsas modelsystem. Sorting in the trans-Golgi network. THE SORTING DEPENDSON SIGNALS IN THE MOLECULESTO BE SORTED

  6. Classical signals: *Sortingof lysosomal enzymes to lysosomes *Basolateral transport in epithelial cells. *Retrograde transport from the Golgi to ER. *Endocytosisof receptorsandother molecules fromthe cell surface – a fraction is sorted to the trans-Golgi network.

  7. Before any basolateral sortingsignalswere identified (1991 ->), it wassuggested that basolateral transport occured by “bulk flow” while transport tothe apicale side – which isthe specialiseddomain in epithelial cells – would require sorting. In 1991 it was published, however, that the transmembrane protein pIgA receptor was transported basolaterallyin a signal dependent manner.

  8. Growth of MDCK epitelial cellson filters. Transfer to glass-disheswith 90 ml of medium for establishmentof confluent cell layers.

  9. Apikalt medium Basolateralt medium

  10. Cytoplasmic domain Baso Api Baso Api Api Api Baso Api Lumenal domain tm

  11. 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 (?)

  12. CT12 CT22 lys-asn-trp-arg-leu-lys-asn-ile-asn-ser-ile-asn-phe-asp-asn-pro-val-tyr-gln-lys-thr-thr-glu-asp- glu-val-his-ile-cys-his-asn-gln-asp-gly-tyr-ser-tyr-pro-ser-arg-gln-met-val-ser-leu- glu-asp-asp-val-ala-COOH CT27 CT37 PM Wild-type Basolateral CT37 Basolateral CT27 Basolateral CT22 Apical CT12 Apical, no endocytosis tyr 18 => ala, wt Basolateral, no endoc. tyr 18 => ala, CT37 Apical, no endoc. tyr 18 => ala, CT27 Apical, no endoc.

  13. Conclusion: There is a tyrosine based basolateral sorting signal in the same region as an endocytosis signal. In addition, there is a second basolateral sorting signal further away from the membrane, outside CT27. This sirting signal has the largest capacity for basolateral sorting (overexpression studies). With this part of the tail alone, a mutation tyr 35 => ala gives 70 % apical receptor. Mutation of both tyr 18 and tyr 35 in wt gives 95 % apical receptor.

  14. YXX (YVEL/YTDI/YXRF) PXXP LL / IL / LEL NPXY YKYSKV YXRF H/R-XXV

  15. Annexin II, Annexin XIIIb

  16. The first proteinsregarded as mediatorsof 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 AP-1B: Epithelia specific AP-3A: Endosome/TGN AP-3B: Neuron-specific (endosome?)

  17. Exchange of material between the TGN, endosomes, lysosomesand the plasmamembrane is mediated largely bycarriers with a dense protein coat regarded to be necessary forselection of content and pinching off from the donor membrane. AP-1, AP-2 and maybe AP-3 (inmammals) may bind clathrin. All 4 complexesare found inArabidopsis, but only AP 1-3 inDrosophila. Many of the subunits are found as closely related isoformes coded by separate genesmaking a large number of combinations possible. Endocytosis

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

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

  20. At least 6 ARFs exist in mammals, 5 are localised to the Golgi-apparatusand 1 to the plasmamembrane. Aktiv membranbundet ARF1-GTP (myristoylated), a tyrosine based signal, andphosfatidylinositol 4,5 bisphosphatearenecessaryto recruit AP-1 clathrin adaptors to membranes. cytoplasmatisk 4 families GEFs with several members Phosphatidylinositidesof the 4-series hasbeen regarded as important for Golgi.

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

  22. All mechanisms for sorting from the TGN are not known *We haveonly discussed proteinswith one transmembrane domain, while many proteins span the membrane several times.These may also be sorted. How? *Some apical proteins, like megalin, havebeen reportedto have signals in the cytoplasmictail (not only luminal signals). *Ubiquitinylation may shift the sorting from TGN to the plasmamembrane towards TGN to lysosomes (the vacuole in yeast) by changing the surrounding circumstances. *Lipids may play a role in sorting in many ways. *What factors are necessary for budding, transport and fusion?

  23. APICAL SORTING IN EPITHELIAL CELLS Glycans: N-glycans, O-glycans, glycosaminoglycans Yes (maybe and no), yes (maybe), yes. GPI-anchors? NOT REALLYLIPID DOMAINS?? Protein motifs for apical sorting: Megalin NPXY. The second of three NPXY motifs is crucial.

  24. 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.

  25. 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.

  26. CS HS

  27. a h c b h c c h c sulfate S apical basol cell Cys/met

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

  29. Could Versican do this?? Versican is reported to bind to sulfated glycolipids*. Could this happen in the Golgi lumen? If yes, is this attachment of any importance for biosynthesis or sorting of Versican? *Miura, Aspberg et al.1999 raft Could the N-term bind other than hyaluronic acid?

  30. WHATISA (GLYCOLIPID) RAFT? Glycolipid- andcholesterol rich domains ina lipidmembraneare associated in a more stable structure than lipids are according to “the fluid mosaic model.” On the cell surface of a “regular” cell, these domainswill have a diameter of 60 - 100 nm. In specialised membranesmay larger areas of the plasmamembrane have raft-characteristics. Example: The apical membraneof epithelial cells (MDCK). Do lipidsand lipid-binding proteinsplay a role in sortingof moleculesthat are transported from the TGN tothe apical membrane?

  31. Caveolins: Proteinswith affinity for specialised lipid-domains. Palmitoylation. Might be necessary for transport of GPI-anchored proteins to the cell surface. GPI-anker: Glycosyl-phosphatidyl-inositol-anchor that might bind proteins toa 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 richin cholesterol. MAL (VIP 17):A protein that seems to mediate apical sortingofseveral cargo proteins in MDCK-cells.

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

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