1. BIOL 200 (Section 921) Lecture # 9, 10 June 29/30, 2006 Readings ECB (2nd ed.) Chapter 15 (Whole chapter): Introduction, pp. 496-501; Protein Sorting pp. 502-504; Protein targeting to the Endoplasmic Reticulum, pp. 505-512; Vesicular Transport, pp. 512 – 516; Secretory Pathway, pp. 516 – 523; Endocytotic Pathway, pp. 523 -529. Questions: 15-3, 15-4, 15-6 to 15-9, 15-12, 15-13, 15-15, 15- 16, 15-18, 15-19, 15-21).

2. Learning Objectives I. Protein Targeting - Nucleus, Mitochondria, Chloroplasts and ER. To explain the nature of signals and sorting To explain the function of coat proteins and the signals carrried by vesicular conponents. To understand the role and importance of the signal recognition particle and the SRP Receptor in the targeting of proteins to the endoplasmic reticulum. To explain how single and multiple pass membrane proteins are inserted into the membrane through use of signal sequences, start transfer sequences and stop transfer sequences. II. Golgi, Vesicle Transport, Endocytosis, Exocytosis. Become familiar with the structure and overall function of the Golgi apparatus Understand how vesicles are formed and targeted. To be able to trace a molecule through each of these pathways describing all of the structures and process through which they pass.

3. pancreas cell Eukaryotes have many membrane bound compartments

4. 15_02_cell_intestine.jpg

6. 01_24_Organelles.jpg How are proteins transported intra- and inter-cellularly?

7. 15_05_import_proteins.jpg

9. 15_06_Signal_sequence.jpg Signal sequences direct proteins to specific organelles

10. Transport of proteins through nuclear pores Active transport – requires GTP hydrolysis Prospective nuclear proteins have a nuclear localization signal (NLS): -Pro- Lys-Lys-Lys-Arg- Lys-Val-

11. 15_10_unfolded_imprt.jpg Protein transport in mitochondria Chaperone proteins pull the proteins across the membranes and refold them Inside the organelles

12. 15_11_ER.jpg Structure of ER: (A) Green fluorescent protein fused to ER resident protein; (B) TEM of a thin section

13. 15_12_pool_ribosomes.jpg Synthesis of cytosolic and ER-bound proteins

14. 15_13_ER_signal_SRP.jpg An ER signal sequence, a signal recognition particle (SRP) and an SRP receptor are required for transport of a protein into the ER

15. 15_14_enters_lumen.jpg A soluble protein enters the ER lumen via the protein translocation channel [Fig. 15-14]

16. 15_15_into_ER_membr.jpg A single-pass transmembrane protein uses two hydrophobic signal sequences: a N-terminal start transfer sequence and a stop transfer sequence

17. 15_16_double_pass.jpg A double-pass ER transmembrane protein uses an internal start-transfer sequence to integrate into the ER membrane [Fig. 15-16] A multipass ER transmembrane protein uses many pairs of start and stop sequences

18. 01_25_endocytosis exoc.jpg Endocytosis Exocytosis (secretion) Vesicular transport - Endocytosis - Exocytosis (secretion)

19. QUIZ: Name the compartments of the secretory and endocytic pathways [Vesicular transport] 3 1 2 4 5 6 Fig. 15-17

20. lysosome endosome Secretory and Endocytic pathways [Fig. 15.24]

21. 15_17_Vesicles_bud.jpg

22. Different vesicle coats drive budding on different compartments [Table 15-4]

23. Table 15-4: Different vesicle coats drive budding on different compartments

24. 15_18_Clathrin_EM.jpg Clathrin protein coat molecules form basketlike cages that help shape membranes into vesicles [Fig. 15.24]

25. Clathrin protein Lattices [Becker]

26. Clathrin triskelions [Becker]

27. 15_19_Clathrin_vesicle.jpg Transport of specific proteins by clathrin-coated vesicles GTP-binding

28. 15_20_SNAREs.jpg The specificity of transport vesicles for their target membranes depends on specific marker proteins, called SNARES

29. 15_21_membr_fusion.jpg SNARE proteins play a central role in membrane fusion [Fig. 15-21]

30. 1.The vesicle is recognized by a coiled-coil tethering protein and a multisubunit tethering complex 2. A RabGTPase bound to the vesicle stimulates formation of a stable complex of one v-SNARE and 3 t-SNARE helices 3. The v-SNARE/t-SNARE interaction promotes Fusion of membranes 4. Binding of the NSF and SNAPs proteins promotes dissociation of SNARE complexes The SNARE hypothesis for transport vesicle targeting and fusion [Becker]

31. What kind of chemical modifications does a protein undergo during vesicular transport?

32. 15_22_glycosylated_ER.jpg Early glycosylation (N-linked) of proteins in the ER [Fig. 15-22]

33. 15_23_Chaperones.jpg Chaperones prevent misfolded or partially assembled Proteins from leaving the ER [Fig. 15-23] Cystic fibrosis: a genetic disease in which a plasma membrane transport protein is slightly misfolded. It would still function if it reached the plasma membrane. But it is retained and degraded in the RER.

34. Functions of Endoplasmic reticulum 1.controls calcium levels in cytoplasm by acting as a calcium store (cell signaling, muscle contraction) 2.site of membrane lipid biosynthesis (sterols and phospholipids). 3.entry point for proteins into the secretory pathway 4.site of post-translational modifications of proteins, eg protein disulfide isomerase forms disulfide bonds here, glycosylation starts here. 5.site of protein folding by chaperone proteins such as BIP (binding protein) which prevent hydrophobic domains of proteins from aggregating and promotes proper folding. 6.quality control checks for proteins (proteins are not exported from ER if they are properly assembled)

35. one Golgi stack [Fig. 15-24]

36. Golgi: stacks of cisternae Each stack=a dictyosome, consists of cisternae (singular, cisterna) CIS TRANS TGN cis/trans polarity Fig. 1-23

37. Models of Golgi Function There are two competing theories: Cisternal progression model: New cisternae form continuously from ER vesicles. Cisternae move through the stack from cis to trans and finally break up into transport vesicles at the trans face. Vesicle transport model: Cisternae remain fixed. Both membrane and content move from the cis to the trans cisternae in transport vesicles.

38. In ER, oligosaccharide added, modified. In Golgi, new sugars added to oligosaccharide Glu Man NGln

39. Protein modifications in Golgi [Fig. 12-6 from Becker] Predict the location of enzymes, galactosyl transferase and sialic acid transferase

40. 15_28_trans_Golgi_net.jpg The regulated and constitutive pathways of exocytosis [Fig. 15-28]

41. Targeting of soluble lysosomal enzymes to endosomes and lysosomes by a Mannose 6-phosphate tag [Becker Fig. 12-9]

42. Protein targeting to lysosomes 1.Proteins destined for lysosomes have a special targeting signal. 2.They are all glycosylated and some of the mannose residues in the attached oligosaccharides are phosphorylated to form mannose-6-phosphate. 3. Mannose-6-phosphate is the targeting signal for lysosomal proteins. 4.The trans Golgi network contains a special mannose-6-phosphate receptor that binds to and results in the concentration of proteins carrying oligosaccharides bearing mannose-6-phosphate into vesicles that are destined for lysosomes.

43. Proteolytic cleavage of proteins as part of processing in secretory granules 1.The later stages in processing of many secreted proteins (e.g. digestive enzymes) involves proteolytic cleavage of a large proprotein to produce a smaller active protein. 2.This typically occurs in secretory granules as they move away from the trans Golgi network. 3. These cleavages are carried out by specific endoproteases (enzymes that cleave polypeptides at sites within the chain).

44. Increased blood glucose level regulates exocytosis of insulin from pancreatic β cells [Fig. 15-29]

46. Endocytic pathways 1.Phagocytosis 2.Pinocytosis 3.Receptor-mediated endocytosis 4.Intracellular digestion in lysosomes

47. 15_30_white_bloodcell.jpg Phagocytosis: A white blood cell ingests a bacterium [Fig. 15-30]

48. 15_31_macrophage.jpg Phagocytosis: A macrophage engulfs two red blood cells [Fig. 15-31]

49. Autophagic digestion of mitochondria [Becker]

50. 15_32_LDL_enters.jpg LDL enters cells via receptor-mediated endocytosis [Fig. 15-32]

51. Receptor mediated endocytosis [Becker Fig. 12-15; ECB Fig. 15-33]

52. 15_34_lysosome.jpg A lysosome contains hydrolytic enzymes and a H+ pump [Fig. 15-34]

53. 15_35_path_lysosome.jpg Different pathways leading to intracellular digestion in lysosomes [15-35]

54. Asbestosis and Silicosis Sharp particles are endocytosed by macrophages. Particles burst lysosomal membranes. Digestive enzymes spill out Another macrophage comes along and the cycle repeats Eventually fibroblasts lay down scar tissue (collagen) to contain damage. Lung now has a hard patch which does not function properly – BLACK LUNG. Also, leads to lung cancer.

55. Experimental techniques for investigating tracking and targeting of proteins and vesicle transport

56. 15_25_methods.jpg Signal sequence is required for protein transport to target organelle

57. 15_26_Temp_mutants.jpg Use of mutants to dissect the protein secretory pathway

58. GFP=green fluorescent protein GFP protein of interest 1. GFP gene fused to gene coding for protein of interest 2. Transform cell with GFP-protein gene fusion. 3.Gene is expressed, targeted, protein functions. 4. Localize Green fluorescence with fluorescent. light microscopy (or confocal) Control-cytoplasmic GFP, i.e. no protein of interest fused onto GFP.

59. PULSE:Cell is exposed to radioactively- labelled nucleotides (green) for 3 minutes. The nucleotides are taken up by the nucleus. CHASE:The cell is exposed to an excess of non-radioactive nucleotides. After five minutes of chase, some radioactive molecules are found in the cytoplasm. 15 MINUTES:Most of the radioactivity has moved from the nucleus to the cytoplasm after 15 minutes of the chase. 30 MINUTES:After 30 minutes, all of the radioactive label is found outside the nucleus. Pulse-chase autoradiography

60. Fig. 8-3 from Karp- Cell and Molecular Biology Pulse chase experiment shows secretory pathway Radioactive proteins labeled in red 3 min20 min 120 min

61. Time course of protein transport Samples are taken after various periods of time and the location of the labeled molecules is identified. (CV) (Z)