Intracellular Transport: Protein Targeting and Vesicle Formation

1. Intracellular Transport Chapter 15 You should review functions of different organelles We already discussed evolution; review Focus will be on: 1) mechanisms of protein targeting and transport to organelles 2) vesicle formation and targeting to organelles and cell surface Questions in this chapter you should be able to answer: Chapter 15: 1 - 8, 9 (all but B),10, 12 - 16, 18,19, 21, 22 Intracellular Transport

2. How did internal membranes arise? Cytosolic membranes Organelle membranes Intracellular Transport

3. Proteins are synthesized in the cytosol… How do they get into appropriate organelles? Three mechanisms: 1) nuclear pores (nucleus) 2) protein translocators (ER, mitochondria, chloroplasts, peroxisomes) 3) vesicular transport (golgi, lysosomes, cell surface) Intracellular Transport

4. How are proteins ‘targeted’ to appropriate membrane? Signal sequences -- N-terminus -- protein surface Intracellular Transport

5. How do proteins pass into chloroplasts and mitochondria? ‘Post-translational’ transmembrane transport N-terminal signal sequence cleaved Post-translational transport Intracellular Transport

6. How do we know that signal peptides are necessary? In vitro translate mRNA for a mitochondrial protein -- radiolabeled (e.g., with 35S) -- assume 55Kd with signal peptide; 53 Kd without signal peptide -- incubate with isolated mitochondria; run through density gradient centrifugation Why does each type of protein occur in the fraction in which it is found? If SDS-PAGE is then performed for the proteins from the two experiments, what would be the molecular weight of the labeled proteins? Intracellular Transport

7. “rough ER” How do proteins pass into ER? ‘Co-translational’ transmembrane transport Signal recognition particle & receptor Protein translocator complex Chaperone proteins aid in folding Intracellular Transport

8. How are transmembrane proteins inserted in the membrane? Single-pass transmembrane proteins -- have a stop-transfer sequence Co-translational transport overview Multi-pass proteins will have multiple ‘start-transfer’ and ‘stop- transfer’ sequences Intracellular Transport

9. What happens to proteins after translation? Covalent modifications e.g. glycosylation acetylation methylation prosthetic groups chemical modifications etc. Preassembly on dolichol-P Intracellular Transport

10. How do proteins pass through nuclear pores? Nuclear localization sequences -- a ‘patch’ on protein surface Pores & nuclear transport receptors -- requires GTP hydrolysis Importin Nucleoporin Question Often nuclear localization signals are not removed from proteins after import into the nucleus. Why? When might they need to be reused? Intracellular Transport

11. What is the most recent model for nuclear import? (see Fig 15-10) Importins (kapβ) nuclear transport receptors that bind Nuclear Localization Sequences Nucleoporins form a gel-like barrier -- have extended phe-gly (FG) repeats Importins cause contraction of nucleoporins Lim, et al. 2007. Nanomechanical Basis of Selective Gating by the Nuclear Pore Complex. Science 318: 640 - 643 Ran / Importin RanGTP allows dissociation & re-export of importin -- GTP  GDP hydrolysis powers export to cytosol Intracellular Transport

12. What is the path of vesicular transport? The endomembrane transport pathway What are the 3 steps of vesicular transport? 1) vesicle budding 2) targeting 3) fusion VSV transport GFP-labeled vesicular stomatitis virus membrane protein from ER to the cell membrane in cultured cells Axonal transport Two-way transport of GFP-labeled protein between Golgi and axonal membrane Notice that there is transport in both anterograde and retrograde directions Intracellular Transport

13. What is the mechanism of vesicle formation? Sorting and packaging are coupled = receptor mediated (endocytosis) Roles of COPI, COPII & Clathrin Clathrin Receptor-mediated Question 15-5 P 506 Intracellular Transport

14. What are the mechanisms of vesicle targeting and fusion? Targetting: Rab proteins & Rab-effectors Fusion & targetting: t- and v-SNARES Energy dependent GTPases Question 15-12 page 522 Intracellular Transport

15. What is the structure and function of the Golgi apparatus? Protein processing & sorting Intracellular Transport

16. ER side anterograde retrograde How does transport occur through the Golgi apparatus? Why does retrograde transport occur? KDEL & ER resident proteins Intracellular Transport

17. What are Regulated and Constitutive secretion? Examples: Insulin secretion Mucus neurotransmitters Regulated secretion & Sea Urchin egg fertilization Intracellular Transport

18. Adapted from Question 15-8 • Fe is transported through blood on protein called transferrin, which can bind to the transferrin receptor on the surface of cells. • Consider these observations: • 1. Fe only binds to transferrin at neutral pH • 2. Transferrin/Fe only binds to its receptor at neutral pH • 3. ‘Empty’ transferrin only binds to its receptor at acidic pH • 4. The transferrin receptor continually cycles from the cell surface to endosomes, and then back to cell surface. • Describe processes & mechanisms that would answer these question: • After being endocytosed into the cell, transferrin and its receptor will move into which compartment? • What happens within this compartment to the FE, transferrin and receptor? • Why aren’t the proteins transported to lysosomes? • What are the advantages of this system? Intracellular Transport

19. What are the functions of and pathways to the lysosome? Vesicular transport from the cell membrane -- endocytosis, phagocytosis vs pinocytosis How are hydrolases targeted to lysosome? -- mannose-6-phosphate Intracellular Transport