3. How many interactions are there? ~6,200 genes ~6,200 proteins x 2-10 interactions/protein ~12,000 - 62,000 interactions Yeast

5. Bait DBD High-throughput yeast two-hybrid BaitPrey DNA binding domain Transcription activation domain + operator or upstream activating sequence Reporter gene transcription Core transcription machinery DBD Act PreyAct

6. Diploid yeast probed with DNA-binding domain- Pcf11 bait fusion protein Haploid yeast cells expressing activation domain- prey fusion proteins High-throughput yeast two-hybrid

10. protein 1 protein 2 protein 3 protein 4 protein 5 protein 6 Bait Tag Affinity column SDS- page Trypsin digest, identify peptides by mass spectrometry High-throughput complex mapping by mass spectrometry

11. 493 bait proteins 3617 “interactions”

12. Bait Tag2 Affinity column1 A variant: tandem affinity purification (TAP) Tag1 Affinity column1 + protease Affinity column2 protein 1 protein 2 protein 3 protein 4 protein 5 protein 6 SDS- page Trypsin digest, identify peptides by mass spectrometry

16. But just how accurate are these high-throughput screens? Four tests: 1. Comparison of mRNA co-expression of interacting partners 2. Comparison of interactions to a test set 3. Comparison of functions of predicted interaction partners 4. Comparison of subcellular localization of interaction partners

17. Scan with laser mRNA fluorescent dyes cells

18. genes experiments expression levels

19. Correlation coefficient between expression vectors True interactions Random Protein Pairs Estimating interaction assay accuracy by assessing mRNA coexpression of partners

20. Estimating interaction assay accuracy by assessing mRNA coexpression of partners

24. At least 1 small-scale expmt >1 independent experiment Paralogs also interact Increasing # of Interaction Sequence Tags >1 independent expmt >2 independent expmt Genome-wide yeast two-hybrid

25. A quantitative measure of pathway reconstruction Cell cycle MAPK signaling pathway Swi4Cdc27 Cell cycle Ubiquitin-mediated proteolysis Systematically test every pair of characterized proteins Jaccard coefficient = # pathways in common / # total pathways Pathways of A Pathways of B pw 1 pw 2 U U pw 1 pw 2  n pairs 1 n = pw 1 pw 2

26. Quality of the observed protein-protein interactions

27. Estimating accuracy with a well-determined reference set of interactions

28. Are interactions between proteins with similar subcellular localization?

29. Summary of tests Authors Method # interactionsMrowkaDeane vonMeringMarcotte Ito et al.Y2H 4081 9% 22% 6-10%~14% Ho et al.MS~3617~10% 1-3% Gavin et al.MS~1440~85% ~10% Uetz et al.Y2H 957 53% 50%~44% Tong et al.synthetic lethal 295~20% >1 independent experiment~2000 85%~30-40%~67% >2 independent experiments 1167 88%~60-70%~73% Co-expression Test set Pathways Estimated True Positive Rate via