Japinder Grewal & Derek Oh 3.6.14 Bio Sci D145

1. JapinderGrewal & Derek Oh3.6.14Bio Sci D145

2. Saccharomyces cerevisiae • Mating only between haploids • MAT locus determines a or α type • Budding yeast cells used • Lots of protein activity • Quick generation times • Easily manipulated

3. Yeast Two-Hybrid System • Eukaryotic transcription factors can form complexes • “Bait” and “Prey” proteins • Expression of reporter gene indicates interaction

4. Methods • 96 ORFs cloned into Gal4 bait and prey plasmids • bait: MATa, ADE2, HIS3 • prey: MATα, URA3, HIS3 • 62 ORFs chosen • 622 = 3,844 mating reactions • 15,523 clones obtained • - 13,754 ISTs obtained

5. Why this method? Any problems? • 13 year old paper, best comprehensive method at the time • PCR-amplification mutations erases interactions • Proper folding of hybrid proteins in plasmids? • Reporter genes sometimes randomly activated • Replaced by direct analysis of protein complexes

6. Results • From the 13,754 ISTs, 4,549 independent two-hybrid interactions left after low quality and redundant IST removal. • 841 of which with 3 or more IST hits (core data) • Compared data with Uetz et al. Also a large- scale two hybrid project.

7. Comparison between genome projects • Used Yeast Proteome Database (YPD). To compare interactions. • Similar rates for known interactions. • Overlapped datasets. Unexpectedly low overlap.

8. What does that mean? • Neither study had >13% of know interactions. • Indicates yeast interactome is larger than previously thought.

9. Genome Two-Hybrid Interaction Map • Using filtered core data (interaction of proteins >3), a protein network was composed. • Large networks in both conventional and two-hybrid analysis- noise ruled out. • Problems -False positive ( false signals, don’t occur in vivo)

10. Tentative function assignment for novel proteins. (Ydr016r’s role in spindle-pole body function) • Molecular mechanisms may have same protein sets, different interactions. • Interaction maps help hypothesize functions of proteins. (editing required)

11. Significance • Shows limitations of two-hybrid analysis generated maps. - Lots of variables between studies (mutation abolishing ORFs, hybrid protein discrepancy, saturation not reached, selection stringency) • Bottom line- multiple or more reliable systems are needed to yield more reliable results.

12. Other techniques • GST (glutathione-S-transferase) pulldown assays • Co-Immunoprecipitation (Co-IP) • Label Transfer Protein Interaction Analysis • BRET assay • FRET - fluorescent resonance energy transfer • Biacore (surface plasmon resonance) • Split-TEV system • Phage display screening

13. Further Reading • Ruderfer, D.M., Pratt, S.C., Seidel, H.S., Kruglyak, L. 2006. Population genomic analysis of outcrossing and recombination in yeast. Nature Genetics. 38:1077-1081 • Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al. (2000)Nature(London)403,623– 627. • http://www.genomeweb.com/node/1109726

14. Any Questions?