Yeast two-hybrid experiments yield information on protein protein interactions
GAL4 Binding Domain
GAL4 Activation Domain
X and Y are two proteins of interest
If X & Y interact then reporter gene is expressed
Invitrogen Yeast 2-Hybrid
Example of 2-Hybrid Analysis
Uetz P. et al., “A Comprehensive Analysis of Protein-Protein Interactions in Saccharomyces cerevisiae” Nature 403:623-627 (2000)
High Throughput Yeast 2 Hybrid Analysis
957 putative interactions
1004 of 6000 predicted proteins involved
Example of 2-Hybrid Analysis
Rain JC. et al., “The protein-protein interaction map of Helicobacter pylori” Nature 409:211-215 (2001)
High Throughput Yeast 2 Hybrid Analysis
261 H. pylori proteins scanned against genome
>1200 putative interactions identified
Connects >45% of the H. pylori proteome
Another Way?
Ho Y, Gruhler A, et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180-183 (2002)
High Throughput Mass Spectral Protein Complex Identification (HMS-PCI)
10% of yeast proteins used as “bait”
3617 associated proteins identified
3 fold higher sensitivity than yeast 2-hybrid
Affinity Pull-down
