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How many interactions are there? Yeast ~6,200 genes ~6,200 proteins x 2-10 interactions/protein ~12,000 - 62,000 interactions High-throughput yeast two-hybrid DBD Bait DNA binding domain + Prey Act Transcription activation domain Core transcription machinery transcription operator or upstream activating sequence Reporter gene High-throughput yeast two-hybrid Haploid yeast cells expressing activation domainprey fusion proteins Diploid yeast probed with DNA-binding domainPcf11 bait fusion protein High-throughput complex mapping by mass spectrometry Tag Bait Affinity column SDSpage protein 1 protein 2 protein 3 Trypsin digest, protein 4 identify peptides by protein 5 mass spectrometry protein 6 493 bait proteins 3617 “interactions” A variant: tandem affinity purification (TAP) Tag1 Tag2 Bait Affinity column2 Affinity column1 SDSpage + protease protein 1 protein 2 protein 3 protein 4 protein 5 protein 6 Trypsin digest, identify peptides by mass spectrometry Affinity column1 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 cells mRNA fluorescent dyes Scan with laser experiments expression levels genes Estimating interaction assay accuracy by assessing mRNA coexpression of partners Random Protein Pairs True interactions Correlation coefficient between expression vectors Estimating interaction assay accuracy by assessing mRNA coexpression of partners >1 independent expmt >2 independent expmt Genome-wide yeast two-hybrid At least 1 small-scale expmt >1 independent experiment Paralogs also interact Increasing # of Interaction Sequence Tags A quantitative measure of pathway reconstruction Swi4 Cdc27 Cell cycle MAPK signaling pathway Cell cycle Ubiquitin-mediated proteolysis Pathways of A Pathways of B pw1 pw2 Jaccard coefficient = # pathways in common / # total pathways S n pw pw1 U 1 <pathway similarity> = n pw2 1 U pw2 pairs Systematically test every pair of characterized proteins Quality of the observed protein-protein interactions Estimating accuracy with a well-determined reference set of interactions Are interactions between proteins with similar subcellular localization? Summary of tests Estimated True Positive Rate via Co-expression Test set Pathways Authors Ito et al. Ho et al. Gavin et al. Uetz et al. Tong et al. Method # interactions Mrowka Deane vonMering Marcotte Y2H 4081 9% 22% 6-10% ~14% MS ~3617 ~10% 1-3% MS ~1440 ~85% ~10% Y2H 957 53% 50% ~44% synthetic lethal 295 ~20% >1 independent experiment >2 independent experiments ~2000 1167 85% 88% ~30-40% ~67% ~60-70% ~73%
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