~50% of the cell volume is in membrane bound organelles

2. ~50% of the cell volume is in membrane bound organelles

3. Each organelle performs specific functions Organelle Main function Structure Organisms Chloroplast photosynthesis double-memb. Plants, protists ER modification, folding single-memb. All eukaryotes of new proteins, lipids Golgi sorting, modification single-memb. All eukaryotes of new proteins Mito energy production double-memb. Most eukaryotes Vacuole storage & housecleaning single-memb. All eukaryotes Nucleus DNA maintenance & double-memb. All eukaryotes RNA transcription

4. General rule:Organelles are generated from the same type of organelle nucleus Nucleus (1) mitochondria Mitochondria (many/ribbon) chloroplasts Chloroplasts (many) endoplasmic reticulum (ER) endoplasmic reticulum (ER) (1) growth division

5. Consequence: during cell division, each daughter cell needs at least one copy of each organelle Different strategies: Nucleus: very precise division and distribution Mitochondria: sufficient numbers, random distribution ER: network is torn apart (perhaps also vesiculation)

6. But, not so clear for Golgi: could be generated from ER For Golgi: two different ideas: 1. Golgi fragments during mitosis, then reforms from fragments 2. Golgi is absorbed into ER during mitosis, then reforms

7. During zygote formation from gamete cells: Egg brings cytosol and all organelles Sperm brings only nucleus All cytoplasmic organelles are derived from mother! (only easily seen with DNA containing organelles, such as mitochondria)

8. Organelles have characteristic shapes Some organelles are more or less spheres e.g. peroxisomes, endosomes, lysosomes, secretory granules Some organelles consist of sheets e.g. Golgi stacks, inner and outer nuclear membrane, outer mitochondrial membrane Some organelles consist of tubules particularly the ER network, mitochondrial inner membrane (cristae), regions of Golgi How is the shape of an organelle brought about and maintained?

9. Membrane structure: lipid bilayer and membrane proteins

10. Each organelle is characterized by a specific set of proteins General rule: every protein is found at only one location (except when en route to its final destination) Most organelles have luminal and membrane proteins (exception: Golgi has no luminal proteins!)

11. Lipids are also localized (although usually not in an absolute manner): Cholesterol: high in plasma membrane, low in the ER (although it is synthesized in the ER!) Phospho- inositides: different forms in different organelles (e.g. PI3P in endosomes, PI4,5P2 in plasma membrane, PI3,5P2 in lysosomes) Cardiolipin: mitochondria

12. How does each organelle receive its specific set of lipids and proteins?Lipid transportProtein transport

13. Lipid transport is still mysterious Questions: How is cholesterol transported? Probably not in vesicles How do mitochondria receive their lipids? They must be received from the ER, but how? How are lipids transported from the outer to the inner mitochondrial membrane? There is no continuity between the two membranes How are lipids flipped from the cytoplasmic leaflet of the ER to the other leaflet of the bilayer?

14. The ER is close to all other organelles in the cell (mitochondria, lysosome, plasma membrane, etc) Could ABC transporter pump lipids from the ER to other membranes? ER Membrane of other organelle ABC (ATP-binding casette) transporter ATPase domain Phospholipid molecules

15. Transport of cholesterol coupled to that of phosphoinositol phosphate (Will Prinz, NIH) Plasma membrane ER membrane phosphatase PI4,5P2 cholesterol Oxysterol binding protein (Osh4p)

16. Protein transportSignals required to direct proteins from the common site of synthesis in the cytosol to their different destinationslike Zip code system

17. NO SIGNAL SEQUENCE Cytoplasmic Nuclear Mitochondrial Plasma membrane Secreted Resident ER and Golgi Endosomes, Lysosomes NUCLEAR LOCALIZATION SEQUENCE MITOCHONDRIAL SIGNAL SEQUENCE ER SIGNAL SEQUENCE

18. The secretory pathway Trans-Golgi Network (TGN) medial ribosome cis trans ER Golgi Plasma membrane

19. Experiments that led to the concept: Pulse-chase experiments (G. Palade) Temperature sensitive VSVG mutant begins to traffic at permissive temp.

20. Two different phases of transport: 1. Translocation (in ER) 2. Vesicular transport

21. Signals must exist to direct proteins: Signal sequences for translocation 2. Sorting or retention signals

22. Translocation across the ER membrane Soluble protein Membrane protein trans-membrane (TM) segment Signal sequence

23. SRP-dependent protein targeting to the ER membrane (in mammals) mRNA ribosome SRP (signal recognition particle) signal sequence of emerging protein translocon SRP receptor

24. Discovery of SRP (Walter & Blobel, 1980) Purified microsomal/rough ER membranes (from dog pancreas) microsomes mRNA + translocation Signal sequence High salt wash mRNA + no translocation Signal sequence Hydrophobic chromatography translocation “SRP” Salt wash

25. SRP contains 6 polypeptides: 11S (~250kDa) SRP SRP 54,68,72 Sucrose gradient SRP 9,14,19 active fraction 11S

26. SRP contains an essential RNA component, 7SL (Walter & Blobel 1982) Noticed that: UV absorbance of SRP at 260 nm >> 280 nm Could it contain nucleic acid???? 260nm absorbance could be reduced by acid hydrolysis-----RNA Is SRP activity sensitive to micrococcal nuclease---------Yes extracted RNA and ran on polyacrylamide gel----- ~260 nts Enzymatic cleavage reaction yield 40 nucleotides at 3’end 7SL

27. Signal Recognition Particle (SRP) contains (6) polypeptides, 1 7SL RNA SRP RNA (7SL RNA) This secondary structure is highly conserved throughout eukaryotes! Zweib et al.

28. SRP-dependent protein targeting to the ER membrane (in mammals) mRNA ribosome SRP (signal recognition particle) signal sequence of emerging protein translocon SRP receptor

29. Identification of the SRP receptor in the ER membrane (Munro, Walter & Blobel, 1981) SRP Salt washed microsomes beads 1% detergent solubilize beads SRP receptor

30. Membrane proteins have different topologies in ER membrane C C N C N cytosol + + + + + + ER _ _ _ _ + + lumen N N cleaved signal sequence C Type I Type II Type III -reverse signal anchor -signal anchor seq. (18-25 apolar) -not cleaved -become ancored in membrane and cause translocation of C-term -cleavable signal seq. (7-15 apolar residues) -SRP dependent -transmembrane anchor stops translocation

31. If only signal sequence: translocation and then secretion (soluble cargo) or plasma membrane (membrane protein) (called default pathway) Note: If protein misfolded in ER- stuck in ER) Sorting and retention signals: Retention in organelle of secretory pathway, e.g. in ER This is often a combination of retention and retrieval

32. Retrieval signals for ER proteins: Luminal proteins: KDEL Membrane proteins: KKXX

33. Identification of KDEL sequence (ER lumen target sequence) (Munro & Pelham, 1987) Some soluble proteins reside in the ER lumen- do they have a sequence in common? looked at sequence of (3) lumenal proteins: grp78, grp94, PDI All (3) have KDEL at their C-terminus Transfect COS cells with grp78, grp94, PDI (+/- KDEL) on each 35S radiolabel protein Immunoprecipitated proteins from cells vs. culture medium Secreted or not? (Lysozyme control + KDEL) sequence is also sufficient for retention

34. Retention/retrieval of ER proteins KDEL Retention/retrieval sequence Golgi low pH KDEL receptor Transport vesicles ER neutral pH

35. If only signal sequence and no ER retention sequence: translocation and then secretion (soluble cargo) or plasma membrane (membrane protein) (called default pathway) Note: If protein misfolded in ER- stuck in ER) Not entirely true, growing list of ER exit COPII packaging signals

36. The secretory pathway Golgi ER Trans-Golgi Network (TGN) medial ribosome cis trans C1 Plasma membrane

37. Budding from ER in COPII coated vesicles Golgi ER Trans-Golgi Network (TGN) medial ribosome cis trans COPII vesicles C1 Mogelsvang et al.

38. COPII Sec13 Sec23 Sec24 Sec31 Sar1 ~60-80nm COPII vesicles COPII coat: important for trafficking from ER to Golgi helps shape membrane (stabilizes curvature) Binds cargo via Sec24 Associates with switch (Sar1/GTPase) so that assembly is reversible

39. COPII Sec13 Sec23 Sec24 Sec31 Sar1 Sec24 binds cargo destined for Golgi- how does it recognize diverse cargoes??? There is no universal ER export signal!

40. (E. Miller & Schekman et al. Cell, 2004) Multiple regions have been found in various proteins required for their export Do all signals bind to the same site of Sec24? Found mutant that no longer binds diacidic motif of Sys1p, Bet1p---- does this mutant still bind other trafficked substrates? VSV-G, Sys1p, Bet1, diacidic motif does not bind mutant Gap1p, Hip1p, Can1p diacidic motif Does Emp24p, Erp1p, Erp2p, ERGIC53 di-hydrophobic motif does not bind mutant ERV41/ERV46 di-hydrophobic motif Does Emp47p tyrosine motif does not bind mutant Sed5 bipartite sorting signal does not bind mutant Bos1p, Sec22p, unknown sequence does not bind mutant Prm8p diphenylalanine motif Does Are their multiple binding sites on Sec24? Sys1p, Bet1p bind to different region of Sec24 than Prm8p and Sec22p

41. How does the COPII complex promote vesicle budding? COPII Sec13 Sec23 Sec24 Sec31 Sar1

42. Acceptor compartment Donor compartment Vesicles have a size of ~60-90nm Questions: 1. How does a vesicle bud? How is cargo concentrated? 2. How is a vesicle targeted? 3. How does a vesicle fuse with its target membrane?

43. Vesicle budding requires coat proteins and the generation of transient membrane curvature COPII coated vesicle ER exit sites: Budding vesicles Sar1p (GTP) CopII High curvature Initial curvature: Sar1p (GTP) Amphipathic helix

44. Model for how COPII generates ER derived vesicles Sec23/Sec24 Binds Sar1 and selects cargo molecules Sar1-GTP Sec23 Sar1-GTP initiates coat formation ER lumen Vesicle (60nm) Sec24 Sec13/31 Induces coat polymerizaton and membrane deformation ER lumen (Lee/Schekman et al.) (Bi/Goldberg et al.) ER lumen

45. How do proteins travel through subsequent Golgi cisternae in stack Golgi Trans-Golgi Network (TGN) medial cis trans ER C1

46. Transport through the Golgi: two models 1. Vesicular transport Cargo moves forward in vesicles Golgi enzymes are stationary 2. Cisternal maturation Golgi enzymes move backwards in vesicles

47. Cisternal maturation model Stable cisternae model Trans cargo Cis

48. Evidence for Cisternal maturation model (Losev/Glick & Matsuura-Tokita/Nakano, Nature 2006) Simple question previously limited by the resolution of light microscope Most eukaryotes Saccharomyces Cerevisiae trans xxxxxxx xxxxxxx xxxxxxx cis So, can follow individual cisternae

49. Evidence for Cisternal maturation model (Losev/Glick & Matsuura-Tokita/Nakano, Nature 2006) SC yeast express GFP-Rer1 (cis Golgi, green) and mRFP-Gos1 (trans Golgi, red) Does a green cisternae turn red?? Or stay green???

50. Golgi: Cisternal maturation model: Questions remain: Can you visualize cargo proteins simultaneously? What happens in cells where the golgi is stacked?