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ER-to-Golgi Transport

ER-to-Golgi Transport. Pathway Maturation De Novo Golgi Biogenesis Mitotic Assembly/Disassembly Post-ER Processing. Pathway Diagram. Sorting, Retention, Retrieval. ER. ERGIC. Golgi. The Golgi is the central processing and sorting station of the secretory pathway. ER. ERGIC. Golgi.

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ER-to-Golgi Transport

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  1. ER-to-Golgi Transport Pathway Maturation De Novo Golgi Biogenesis Mitotic Assembly/Disassembly Post-ER Processing

  2. Pathway Diagram

  3. Sorting, Retention, Retrieval

  4. ER ERGIC Golgi The Golgi is the central processing and sorting station of the secretory pathway ER ERGIC Golgi

  5. Live Imaging of VSVG-GFP Transport

  6. COPII Mediates ER Export

  7. Homotypic fusion COPII vesicles may form ERGIC/VTCs

  8. VTC morphology

  9. ERGIC/VTC moves along microtubules - +

  10. The Golgi maintains its complex structure in the presence of continuous membrane traffic trans medial cis

  11. 3D View of Golgi Stack

  12. By what mode does Golgi traffic occur?

  13. Vesicle & Maturation Models

  14. Enzymes in COPI? Table II. Relative lateral distribution of Golgi proteins and VSV-G within the Golgi complex Golgi stack (cisternae) Lateral rims (uncoated) Coated buds and vesicles Man II 68.9 ± 3.9 13.6 ± 2.7 17.5 ± 2.8 Giantin 58.2 ± 2.6 18.3 ± 2.8 23.5 ± 2.7 KDELr 35.8 ± 4.3 8.9 ± 2.0 55.3 ± 4.5 rBet1 25.6 ± 4.9 15.7 ± 4.0 58.7 ± 5.6 VSV-Gts045–GFP (20 min, 32°C) anti-GFP 93.1 ± 1.4 4.3 ± 1.3 2.6 ± 0.6 anti–VSV-G–lum 94.1 ± 0.8 3.4 ± 0.6 2.5 ± 0.5 ------------------------------------------------------------------------ Numbers represent the percentages (mean ± SEM) of the total labeling over the distinct membrane categories and were obtained by analyzing 50 Golgi complexes for each antibody.

  15. Enzymes not in COPI?

  16. COPI Vesicles Concentrate Enzymes not Cargo ------------------------------------------------------------------------ Markers Amount of vesicles (% of starting membranes) ------------------------------------------------------------------------ GTP GTPS arf-1 Q71L ------------------------------------------------------------------------ Golgi enzymes Mann II 13.2 ± 1.3 6.1 ± 1.4 4.2 ± 1.1 NAGT I 25.8 ± 2.7 3.1 ± 1.0 3.0 ± 0.8 GalT 11.0 ± 2.0 1.7 ± 0.4 1.8 ± 0.4 Anterograde cargo pIgR  4.6 ± 1.0 3.6 ± 1.4 3.3 ± 1.1 Phospholipids  2.7 ± 0.7 3.1 ± 0.7 2.5 ± 0.8 ------------------------------------------------------------------------

  17. How is the Golgi Formed?

  18. Trafficking may be sufficient for Golgi self-assembly Golgi ERGIC cis medial trans Trans Golgi Network Homotypic fusion MT COP I COP I COP I ER COP II

  19. Can the Golgi self-assemble? Idea: Use a reversible ER export block -Brefeldin A to collapse Golgi -H89 to block ER export and collapse ERGIC

  20. Collapse of matrix & non-matrix markers into ER

  21. GM130 co-fractionates with ER membranes Golgi ER untreated BFA H89

  22. min after H89 washout 0’ 4’ Re-emergence of the Golgi from the ER GM130 10’ 30’

  23. Reassembled Golgi membranes are stacked BFA H89 30’ washout

  24. Early compartmentalization of Golgi membranes after ER exit 5’ Giantin GPP130

  25. Initial Stages in “de novo” Golgi biogenesis

  26. De novo biogenesis continued

  27. Transport paths & consequences of their regulation Mitotic: vesicles=>block in docking Osmotic:ER=>ER export block

  28. M-phase Golgi Vesiculation

  29. Tethered vesicle transport: inhibited at M-phase?

  30. Anti-p115 injection induces dispersion of the Golgi in vivo

  31. Blocking p115, but not giantin or GM130, causes vesiculation

  32. What are the essential p115 interactions? (i.e. candidates for regulation at mitosis) acidic tail head domain coiled-coil domain Rab1 Giantin GM130 PLCg1 SNAREs phosphorylation site GBF1

  33. Beauty is in the RNAi of the Beholder

  34. 100 80 60 40 mock 24 h 48 h 72 h 96 h 20 0 mock 96 h p115 siRNA induces time-dependent depletion of p115 GPP130 p115 p115 mock %intact p115 levels siRNA

  35. siRNA-induced p115 depletion results in Golgi fragmentation p115 GPP130 mock siRNA

  36. 20 40 60 80 100% f +p115 Mismatched p115 rescues siRNA-induced Golgi fragmentation inj. marker p115 GalNac-T2 % injected cells with intact Golgi

  37. O The giantin/GM130-binding domain is NOT required for Golgi biogenesis in vivo WT S941A Dtail Acidic Tail 20 40 60 80 100% Targeted? + WT + S941A + Dtail % expressing cells with intact Golgi

  38. O WT Dcc1 The SNARE domain of p115 is required for Golgi biogenesis in vivo cc1 + + + + + ? + Targeted?

  39. Working model for mechanism of action of p115 recruitment of p115 by Rab1 binding to t-SNARE catalysis of SNARE pairing p115 v-SNARE Rab1 t-SNARE => dissect SNARE mechanism w/ further mutagenesis => test other p115 interactions-> distinct phenotypes?

  40. rbet1 syntaxin5 membrin sec22 Sub-regions of the cc1 domain show homology to distinct SNAREs rbet1 cc1 core complex domain syntaxin5 membrin p115 homology region sec22

  41. Part 1 Post-ER Processing Modification of carbohydrate side-chains Proteolytic activation of precursors

  42. Compartmentalization of processing reactions

  43. Branched carbohydrate

  44. Evidence for enzyme compartmentalization

  45. Processing Reactions endoH sensitive endoH resistant cis trans

  46. Example of proteolytic activation

  47. Proton pumps reduce lumenal pH

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