NATURE |VOL 403 | 10 FEBRUARY 2000

1. A comprehensive analysis ofprotein-protein interactionsin Saccharomyces cerevisiaePeter Uetz, Loic Giot, Gerard Cagney et.al. NATURE |VOL 403 | 10 FEBRUARY 2000

2. Saccharomyces cerevisiae is a species of buddingyeast. It is perhaps the most useful yeast owing to its use since ancient times in baking and brewing. • It is one of the most intensively studied eukaryoticmodel organisms in molecular and cell biology, much likeE.coli as the model prokarote. • Saccharomyces cerevisiae cells are round to ovoid, 5–10 micrometres in diameter. It reproduces by a division process known as budding http://en.wikipedia.org/wiki/Image:S_cerevisiae_under_DIC_microscopy.jpg

3. S. cerevisiae was the first eukaryoticgenome that was completely sequenced. (April 1996) • The genome is composed of about 13,000,000bp and 6,275 genes, although only about 5,800 of these are believed to be true functional genes.

4. Yeast Two-Hybrid Analysis • Two-hybrid screening is a technique used to discover protein-protein interactions by testing for physical interactions (such as binding) between two proteins . • The most common screening approach is the yeast two-hybrid assay. The method is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation. Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-binding domain fused to protein X and the other consisting of the GAL4 activation domain fused to protein Y, are constructed and introduced into yeast. Interaction between proteins X and Y leads to the transcriptional activation of a reporter gene containing a binding site for GAL4.

5. Yeast Two-Hybrid Analysis • 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

6. Approaches used: Two large-scale yeast two-hybrid screens were used to identify protein-protein interactions between full-length openreading frames predicted from the Saccharomyces cerevisiae genome sequence.

7. Method 1: A protein array of activation-domain hybrids • Can some one briefly describe the protein array screens ?

8. A protein array of activation-domain hybrids MATa strain DNA-binding domain 192 hybrids MAT αstrain activation-domain 6000 ORFs x mated on YEPDplates for 2-3 days two-hybrid selection on medium without leucine, tryptophan and histidine supplemented with 3mM 3-amino-1,2,4-triazole Diploids were selected on medium without leucine and tryptophan 2-3 days of further growth. scored after two weeks of growth at 30 ℃

9. Figure 1 The two-hybrid assay carried out by screening a protein array • What does the medium in Figure 1a select for? • What does the medium in Figure 1b select for?

10. Method 2 High-throughput screens of an activation-domain library • Can some one briefly describe the high-throughput library screens ?

11. Method 2 High-throughput screens of an activation-domain library MATa strain Gal4 DNA-binding domain fusion x MATαstrain activation-domain library Mated on 96-well filter plates Diploids containing potential interactors wereselected for 4 days at30 ℃ on medium lacking leucine, tryptophan and uracil, andimultaneously screened for lacZ expression by the addition of X-gal. Incubated overnight onrectangular YPAD solid medium plates. Collected cells from the mating mixes with sterile water Up to 12 blue colonies were picked per mating

12. The array screens and the library screens gave different data sets ?

13. 45% of the 192 proteins used in the arrayscreens yielded interactions, compared to 8% of the 5,345 potentialORFs in the library screens. ------attributableto the nonrandom choice of proteins for the array screens • Thelibrary approach permits a much higher throughput, the arrayscreens generate more candidate interactors. ------partially attributed to thestringency of the selection procedures; the His selection for 14 daysin the array screens was less stringent than the Ura selection for4 days in the library screens. ------ The array may facilitate the detection ofinteractions that result in very low reporter gene activation. • But array screens are much more labour- and material-intensive, andrequire several hours of robot time per screen, thus severely limitingthe number of screens that can be performed.

15. Sum of Table 3 • Representative proteins from 41 of the 43 yeast protein database (YPD)categories were identifed in the screens. • Of the 1,004active proteins, 412 fell into the unclassified category. • These 412proteins yielded 509 distinct interactions, of which 164 (32%) werebetween proteins with no functional classification. • This observationindicates that there may be a significant number of as yet undiscoveredpathways and/or complexes that can be identified usingsystematic approaches.

16. Results from both approaches were alsocompared with a compilation of literature-cited interactions(Table 2). • Only a subset of previously describedinteractions has been detected in their screens, why ???????

17. Figure 2 Data analysis software. What does Fig. 2 tell us?

18. Figure 3 Expanded pathways shown using the software as described in the text What can we get from Fig.3?

19. Summary • Two large-scale yeast two-hybrid screens(the array screens and the library screens) were undertaken to identify protein-protein interactions between full-length openreading frames predicted from the Saccharomyces cerevisiae genome sequence. • These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. • These data revealinteractions that place functionally unclassified proteins in a biological context, interactions between proteins involved in thesame biological function, and interactions that link biological functions together into larger cellular processes.

20. Discussion • What are the advantages and disadvantages of the yeast two-hybrid analysis ?

21. Discussion • How to overcome these limitations?

22. Discussion • How can we use other methods we discussed before, e.g., mass spectrometry, co-precipitation, biomolecules, etc. to verify the data of the yeast two-hybrid analysis ?