1. A road map for cell biology: Why studying large protein complexes is crucial at this time David Drubin, UC Berkeley

2. Why study large complexes? Proteins typically function in association with other proteins. Protein complexes are important for virtually every biological process and most diseases. Genome sequences identify tens of thousands of genes: linking these to 200-300 core biological processes will make their study manageable. Recently developed and/or improved technologies and methodologies make studies of large complexes more feasible and informative.

3. A brief tour of some protein complexes and their biological and biomedical importance (from the Alberts et al. textbook)

4. Nucleosome

5. DNA Polymerase

6. Nuclear pores

7. Ribosome

8. Proteosome

9. Cell Cycle: Short-lived complexes

10. COPII-coated vesicle

11. MT motor - vesicle

12. Enzyme (Aspartate transcarbamoylase) ; two conformations

13. Red blood cell skeleton

14. Signal transduction complex

15. Many new and improved approaches for studies of protein complexes are now available Using combinations of these approaches makes the most of their complementary features These approaches can be used in targeted or genome-wide studies Approaches to studies of protein complexes

16. Structural Proteomics Biological function mechanism regulation Protein localization Biochemical activities Protein structure Expression studies Protein-protein interactions Genetic interactions Mutant/RNAi/ Chemical phenotypes Post-translational modifications

17. Features of genome-wide, non-targeted “big science” *Efficiency *Unbiased *Patterns can emerge that only are evident when analyzing large datasets *Validation and follow-up, quality control Pros: Cons:

18. Features of targeted studies “small science” *Science is an iterative process -much can be missed with one pass *Appreciation of vital subtleties of a system, knowing where to look and what to look for Cons: *Less efficient Pros: *Tools and insights for validation and quality control

19. Non-Targeted “Big Science” Targeted “small science”

20. Technologists Biologists

21. Mass spectrometry is having a revolutionary effect on identification of subunits of protein complexes and their post-translational modifications Mass Spectrometry

22. The Budding Yeast Kinetochore

23. (Scott Anderson and John Yates)

24. Molecular Analysis of Kinetochore Composition and Organization 3 novel kinetochore sub-complexes defined 28 kinetochore proteins purified (75% of total) 5 novel kinetochore proteins identified

25. Light microscopy can verify the in vivo relevance of proteomically observed associations, and provide valuable information about dynamics Light microscopy

26. Dad3p and Dad4p Localize to Spindles and Kinetochores Spindle (Intact Cells) Kinetochore (Chromosome Spreads)

27. Tandem Mass Spectrometry Mapping of Phosphorylation Sites Dam1p Complex - Ndc80p Complex - Ctf19p Complex - Ipl1p Complex - in vivo Phosphorylation Sites 13 1 4 0 Ipl1p Targets 6 1 3 0

28. Genetics can verify in vivo relevance of post- translational modifications, associations, etc. Genetics

29. dam1 (S to D) Mutants Suppress ipl1-2 dam1 (S to D) ipl1-2 dam1 (S to D) Wild Type ipl1-2

30. The current demand for mass spectrometry outstrips the existing capacity Cell cycle stages Response to stimuli and perturbation Affects of various mutants

31. Clone John Yates

32. Two hybrid Arrays Yeast Resource Center (Stan Fields) All yeast ORFs on 16 plates

35. HIGH QUALITY datasets from large-scale, genome-wide analyses such as two-hybrid screens, mass spec of complexes, etc., can greatly accelerate biological research. Datasets from non-targeted proteomics

36. Yeast WASp

37. GFP, its spectral variants, and DsRed are driving the need for advanced imaging systems to study protein complexes in vivo -High speed multi-color analysis -FRET -FRAP -Automated image acquisition and analysis Light microscopy is critical for determining where and when proteins associate in complexes

38. Endocytic protein interactions

39. Why doesn’t the whole cytoplasm precipitate? Interactions are regulated in space and time!

40. Small molecule inhibitors can be powerful tools to switch on and off associations within protein complexes in vivo Chemical Biology (Genetics)

41. Summary Protein complexes are involved in virtually every cellular process and disease Complete genome sequences combined with availability of new technologies make studies of protein complexes crucial at this time The focus of all such studies should be on illuminating biological and disease mechanisms

42. Acknowledgements Yeast Resource Center (John Yates, Stan Fields, Trisha Davis) Kevan Shokat (UCSF) Barnes and Drubin labs (UCB)