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BIOL 200 (Section 921) Lecture # 9, 10 June 29/30, 2006

BIOL 200 (Section 921) Lecture # 9, 10 June 29/30, 2006. Readings

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BIOL 200 (Section 921) Lecture # 9, 10 June 29/30, 2006

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  1. BIOL 200 (Section 921)Lecture # 9, 10June 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. Eukaryotes have many membrane bound compartments pancreas cell

  4. 15_02_cell_intestine.jpg

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

  6. 15_05_import_proteins.jpg

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

  8. 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-

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

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

  11. 15_12_pool_ribosomes.jpg Synthesis of cytosolic and ER-bound proteins

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

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

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

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

  16. 01_25_endocytosis exoc.jpg Endocytosis Exocytosis (secretion) Vesicular transport - Endocytosis - Exocytosis (secretion)

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

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

  19. 15_17_Vesicles_bud.jpg

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

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

  22. 15_18_Clathrin_EM.jpg Clathrin protein coat molecules form basketlike cages that help shape membranes into vesicles [Fig. 15.24]

  23. Clathrin protein Lattices [Becker]

  24. Clathrin triskelions [Becker]

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

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

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

  28. 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 4. Binding of the NSF and SNAPs proteins promotes dissociation of SNARE complexes 3. The v-SNARE/t-SNARE interaction promotes Fusion of membranes The SNARE hypothesis for transport vesicle targeting and fusion [Becker]

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

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

  31. Chaperones prevent misfolded or partially assembled Proteins from leaving the ER [Fig. 15-23] 15_23_Chaperones.jpg 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.

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

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

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

  35. 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.

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

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

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

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

  40. Protein targeting to lysosomes • Proteins destined for lysosomes have a special targeting signal. • They are all glycosylated and some of the mannose residues in the attached oligosaccharides are phosphorylated to form mannose-6-phosphate. • Mannose-6-phosphate is the targeting signal for lysosomal proteins. • 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.

  41. Proteolytic cleavage of proteins as part of processing in secretory granules • 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. • This typically occurs in secretory granules as they move away from the trans Golgi network. • These cleavages are carried out by specific endoproteases (enzymes that cleave polypeptides at sites within the chain).

  42. Increased blood glucose level regulates exocytosis of insulin from pancreatic βcells [Fig. 15-29]

  43. Endocytic pathways • Phagocytosis • Pinocytosis • Receptor-mediated endocytosis • Intracellular digestion in lysosomes

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

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

  46. Autophagic digestion of mitochondria [Becker]

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

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