1. The Yeast Resource Center An NCRR Biomedical Technology Resource Center http://depts.washington.edu/~yeastrc

2. Focus Technologies that extract information about macromolecular interactions from the genome sequence. Regulation Dynamics

3. The people The technologies The YRC is a consortium of five groups and five technologies –Aebersoldmass spectrometry –Yatesmass spectrometry –Fieldstwo-hybrid –Bakerprotein struct. prediction –Muller/Davismicroscopy and FRET –Davis/Bakerinformatics

4. A P41 center has three missions Technology development Collaboration Dissemination and Training

5. By the numbers (for 2002) 51 publications Technology development: –19 projects Collaborations: –132 collaborators, 80 projects Dissemination and Training: –130 individuals 26 states, 12 countries

6. Unknown ORFs 105 unknown essential ORFs Integrate all our technologies Find a balance between quantity of data and quality of data

7. Analysis of high throughput data von Mering et al., 2002

8. Unknown essential ORFS Tag with TAP tag and YFP tag Purify each with TAP tag Identify copurifying proteins by mass spec Localize in living cells Two-hybrid analysis Advanced homology searches Protein structure prediction

9. * * * * 212 43 56 97 66 36 Standards YGR046W (44kD) YFR003C (18kD) YGR002C (55kD) YJR013W (35kD) YKR079C (97kD) Spc97p * * * * * * 212 43 56 97 66 36 YHR085W (38kD) YKR038C (47 kD) Standards YGR024C (28kD) YNL158W (22kD) Spc97p YOL034w (126 kD) Optimization of purification with TAP tag Old method Graczyk, Sundin, Fox and Davis New method

10. Summary of purification 20 are part of a complex –15 different complexes 28 purify by themselves 22 could not be tagged 26 were inactivated by tag 9 were not produced at detectable levels in logarithmic cultures

11. Integration of technologies builds the story New complex, ribosomal recycling –All four members are essential unknowns –One localizes to nucleolus, three others killed by tag –Two-hybrid confirms mass spec –Homology searching finds homology of one to ribosomal recycling factor

12. Integration of technologies builds the story An ER protein that purifies alone –Mass spec finds only that protein –Localizes to ER –Two-hybrid identifies a collection of membrane proteins –Homology searching identifies homology to mannosyltransferases

13. Integration of technologies builds the story YKR079c-TAP copurifies with 2 proteins. But localization reveals they are likely to form two complexes of two proteins. YKR079cNUC1YMR099c

14. Lessons Mass spec is extremely powerful Two-hybrid expands the story and provides critical information for proteins not in complexes Localization is fast and easy Great deal of information to be had merely by advanced homology searching Protein structure prediction is underway

15. Recommendations Think in terms of optimal throughput which strikes a balance between quantity and quality. More mass spec capacity is needed and the technology is not yet readily disseminated. Promote the integration of technologies. Increase capacity and speed of advanced homology searches.

16. Yeast Resource Center John Yates –Scott Anderson –Hayes McDonald Ruedi Aebersold –Rick Newitt –Eugene Yi Stan Fields –Tony Hazbun –Jennifer Aranda Eric Muller –Brian Snydsman Trisha Davis –Bethany Fox –Beth Graczyk David Baker –Lars Malmström –Bill Schief Informatics –Mike Riffle