1. The Yeast Two-Hybrid System Anne C. Luebke

2. What is the yeast two-hybrid system used for? n Identifies novel protein-protein interactions n Can identify protein cascades n Identifies mutations that affect protein- protein binding n Can identify interfering proteins in known interactions (Reverse Two-Hybrid System)

3. How does it work? n Uses yeast as a model for eukaryotic protein interactions n A library is screened or a protein is characterized using a bait construct n Interactions are identified by the transcription of reporter genes n Positives are selected using differential media

4. The Model DNA-Binding Domain Bait ProteinPrey Protein Transcription Activating Region Reporter Gene DNA-Binding Site

5. Steps to Screen a Library n Create the Bait Plasmid Construct from the gene of interest and the DNA binding domain of Gal4 or LexA or other suitable domain n Transform with the bait construct a yeast strain lacking the promoter for the reporter genes and select for transformed yeast n Transform the yeast again with the library plasmids n Select for interaction

6. Sequence analysis n Isolate plasmid from yeast and transform E. coli n Purify plasmid from E. coli and sequence n Blast sequence against database for known proteins or construct a possible protein sequence from the DNA sequence and compare to other proteins

7. Reporter Genes n LacZ reporter - Blue/White Screening n HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity) n LEU2 reporter - Screen on Leu+ media n ADE2 reporter - Screen on Ade+ media n URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)

8. Plasmid Constructs n Plasmids are constructed with the Gal4 DNA binding domain (or other suitable domain) in front of a Multiple Cloning Site (MCS) n The plasmid contains genes that can be used for selection such as Amp, Leu2, Ura3, or Trp1

9. Sample Plasmid From Golemis Lab Homepage

10. False Positives n False positives are the largest problem with the yeast two-hybrid system n Can be caused by: n Non-specific binding of the prey n Ability to induce transcription without interaction with the bait (Majority of false positives)

11. Elimination of False Positives n Sequence Analysis n Plasmid Loss Assays n Retransformation of both strain with bait plasmid and strain without bait plasmid n Test for interaction with an unrelated protein as bait n Two (or more) step selections

12. Advantages n Immediate availability of the cloned gene of the interacting protein n Only a single plasmid construction is required n Interactions are detected in vivo n Weak, transient interactions can be detected n Can accumulate a weak signal over time

13. Examples of Uses of the Yeast Two-Hybrid System n Identification of caspase substrates n Interaction of Calmodulin and L-Isoaspartyl Methyltransferase n Genetic characterization of mutations in E2F1 n Peptide hormone-receptor interactions n Pha-4 interactions in C. elegans

14. References n Bartel, Paul, C. Chien, R. Sternglanz, S. Fields. “Elimination of False Positives that Arise in Using the Two-Hybrid System.” Biotechniques (1993) Vol. 14, no. 6, p. 920-924. n Chien, Cheng-ting, P. Bartel, R. Sternglanz, S. Fields. “The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest.” Proc. Natl. Acad. Sci. USA (1991) Vol. 88, p. 9578-9582. n Fields, Stanley, O. Song. "A novel genetic system to detect protein-protein interactions." Nature (1989) Vol. 340, p.245-246. n James, Philip, J. Halladay, E. Craig. "Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast." Genetics (1996) Vol. 144, p. 1425-1436. n Kamada, S, H. Kusano, H. Fujita, M. Ohtsu, R. Koya, N. Kuzumaki, Y. Tsujimoto. "A cloning method for caspase substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin." Proc. Natl. Acad. Sci. USA (1998) Vol 95, p. 8532-8537. n O'Connor, Mirriam, C. O'Connor. "Complex Interactions of the Protein L-Isoaspartyl Methyltransferase and Calmodulin Revealed with the Yeast Two-hybrid System." The Journal of Biological Chemistry (1998) Vol. 273, p. 12909-12913. n Staudinger, Jeff, J. Zhou, R. Burgess, S. Elledge, E. Olson. "PICK1: A Perinuclear Binding Protein and Substrate for Protein Kinase C Isolated by the Yeast Two-Hybrid System." The Journal of Cell Biology (1995) Vol. 128, p. 263- 271.

15. References continued n Vidal, Marc, P. Braun, E. Chen, J. Boeke, E. Harlow. "Genetic Characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system." Proc. Natl. Acad. Sci. USA (1996) Vol. 93, p. 10321-10326. n White, Michael. "The yeast two-hybrid system: Forward and reverse." Proc. Natl. Acad. Sci. USA (1996) Vol 93, p. 10001-10003. n Zhu, Jianwei, C. Kahn. "Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system." Proc. Natl. Acad. Sci. USA (1997) Vol. 94, p. 13063-13068. n Lab of Erica Golemis http://www.fccc.edu/research/labs/golemis/EG_homepage.html n Special thanks to Dr. Susan Mango and the University of Utah