1. 01.26.11 Lecture 6 - Intracellular compartments and transport I

2. Intracellular transport and compartments 1. Protein sorting: How proteins get to their appropriate destinations within the cell 2. Vesicular transport: How vesicles shuttle proteins and membranes between cellular compartments

3. Primary functions of the compartments within a cell

4. Relative cellular volumes of the major membranous organelles

5. The evolution of organelles: nuclear membranes and ER

6. The evolution of organelles: mitochondria

7. Organelles import proteins by three distinct mechanisms 1. Transport from the cytoplasm into the nucleus through nuclear pores 2. Transport from the cytoplasm to organelles by protein translocators in the membrane 3. Transport from ER to other organelles occurs via vesicles

8. Signal sequences target proteins to their destinations

9. Signal sequences are necessary and sufficient for protein targeting

10. Mechanism 1: proteins enter the nucleus via nuclear pores The nuclear envelope is a double membrane Contiguous with the ER - both compartments share the same lumen Perforated by nuclear pores

11. The nuclear pore complex (NPC) is a selective molecular gate Composed of ~100 different proteins Small, water-soluble molecules pass freely, macromolecules must carry appropriate signal

12. NPCs actively transport proteins bound for the nucleus 1. Proteins bind to nuclear transport receptors 2. Complex is guided to the pore by filaments 3. Pore opens, receptor + protein are transported in (uses GTP) 4. Receptor is shuttled back into the cytoplasm

13. GTP hydrolysis provides the energy that drives nuclear transport

14. The nuclear envelope disassembles and reforms during each cell division

15. Mechanism 2: protein translocation from cytoplasm to organelle Proteins moving from the cytosol into the ER, mitochondria, chloroplasts, or peroxisomes Protein movement is mediated by specialized proteins termed protein translocators Unlike passage through nuclear pores, translocation requires unfolding or co- translational transport

16. Proteins are unfolded during translocation into mitochondria

17. The ER is a network of membrane sheets & tubules

18. Active ribosomes may be in the cytosol or associated with the ER

19. Ribosomes are directed to the ER by the SRP and ER signal

20. Soluble proteins cross the ER membrane into the lumen

22. Integration of transmembrane proteins into the ER membrane

23. Multi-pass proteins use internal start- transfer sequences

24. Mechanism 3: vesicular transport

25. Transport vesicles Continually bud off from and fuse to other membrane compartments producing a constant flux of material Carry soluble proteins (in the lumen) and lipids & membrane proteins (in the bilayer) between compartments Are transported along microtubules by motor proteins

26. Vesicle budding is driven by assembly of a protein coat

27. Clathrin-coated vesicles transport selected cargo molecules

28. Complexes of clathrin form a basket around vesicles and help them to pinch from membranes

29. Step 1 Cargo molecules (red) bind to transmembrane cargo receptors Cytoplasmic domains of receptors bind to adaptin (light green)which recruits clathrin Clathrin clusters cargo/receptor/adaptin complexes and induces curvature to the membrane - clathrin-coated pit

30. Step 2 Additional clathrin molecules bind - increasing curvature Dynamin assembles a ring around each clathrin- coated pit

31. Step 3 Dynamin rings constrict to “pinch” the membrane off Dynamin is a GTPase and used the energy released from GTP hydrolysis to power this reaction

32. Step 4 The free vesicle sheds its coat of adaptin and clathrin Vesicles are transported to their destination on microtubules

33. Clathrin-coated vesicles transport selected cargo molecules

35. Animation of clathrin assembly and disassembly around an endocytic vesicle

36. SNAREs are proteins that target vesicles to specific compartments t-SNARES are on target compartments v-SNAREs are on vesicles

37. SNARE proteins are important for membrane fusion v-SNAREs and t-SNAREs bind tightly Complexes bring the two membranes together to promote fusion