1. Chemical reactions in cells need to be isolated. Enzymes work in complexes, spatial distribution in cytosol, nucleus Confinement of reactions in organelle compartments.

2. Mitochondria 22% vol. ER 12 Nucleus 6 Golgi 3 Peroxisomes 1 Lysozomes 1 Endosomes 1

3. Organelles can be partially purified by centrifugation Mitochondria Nuclei Plastids Glycogen Microsomes (ER)- remember this- we come back to it later

4. Surface to volume ratio problem No problem for prokaryotes but eukaryotes (1000+ times larger) needed to evolve compartments How?

5. This way for the nucleus and endomembrane system

6. This way for mitochondria and chloroplasts

7. Compartmentalization solved one problem and created another: How is a cell with all its organelles recreated? How are proteins and lipids moved around?

8. 3 ways occur

9. Proteins carry their own zip codes: signal sequences and patches

10. Proteins enter the nucleus through pores

11. Side view Face-on Water filled pore, 100’s of subunits comprise this elaborate pore

12. Nuclear transport is an active process. Requires GTP hydrolysis Transport occurs in the folded state

13. But transport through other membranes requires UNfolding Cellular postmen recognize the zip codes (signal sequences)

14. Plant cell w/ GFP ER Dog pancreas cell- lots of secretion here Proteins in the endomembrane system or secreted are first directed to the ER

15. Single ribosome pool serves both free translation and co-translation

16. Many components are needed for co-translational import.

17. How do we know? In vitro experiments based on reconstitution of the components

18. Translocation of secretory proteins across the ER Signal Sequence Microsomes required Signal sequence cleaved Signal peptidase in lumen SS= charge/hydrophob/charged ca. 70 amino acids A few are different (alpha factor)

19. 40 30 In vitro systems plus genetics (next lecture) have unraveled the sequence.

20. The SRP and SRP receptor is a GTPase

21. Cellular addresses of proteins reside in the primary and secondary structure Cellular postmen recognize these addresses by direct interaction.

23. Secretory proteins move from the rough ER lumen through the Golgi and then to the cell surface. 1.Transport vesicles 2.Cisternal progression 3.Regulated and constitutive secretion 4.Lysozomes

24. George Palade 1974 Nobel Prize Medicine Randy Sheckman

25. Temperature sensitive invertase mutants do not secrete invertase at the nonpermissive temperature. Pulse chase experiments were used to identify were secretion was blocked. Analysis of such mutants revealed 5 classes. Cloned genes (~30) indicated molecular components of the secretory pathway

36. Vesicular traffic extends to and from the plasma membrane. New proteins of the ER are delivered to the Golgi apparatus and to the cell surface by vesicles.

37. Protein modification begins in the ER lumen.

38. Misfolded proteins are degraded (quality control). E.g. a cftr mutation causes CFTR not to be delivered to the plasma membrane

39. Fibroblast Fluorescent antibody Stains the Golgi