1. Assigning Function to Yeast Proteins By Integration of Technologies Hazbun, TR et al. (2003) Mol. Cell 12, 1353-1365. Kate Drahos 23 April 2004

2. Overview Although the yeast genome is fully sequenced, 1/3 of ORFs are not functionally annotated Large-scale studies provide incomplete data Small-scale studies are quite directed and provide biased information Further analysis is required for complete functional annotation of the yeast genome

3. Goals Set of 100 essential ORFs whose functions are unknown Protein products of ORFs subjected to a set of four different experimental approaches 1) Tandem Affinity Purification & MudPIT 2) Yeast Two-Hybrid Screen 3) Fluorescence Localization 4) Protein Structure Prediction Aim to assign roles to these proteins based on standardized GO categories

4. Tandem Affinity Purification TAP used to purify target ORFs and copurifying proteins TAP allows for native expression levels of the tagged protein and purification of unknown protein complexes (adapted from Rigaut et al., 1999) Proteins visualized by SDS-PAGE and silver staining

5. Multidimensional Protein Identification Technology MudPIT developed for analysis and identification of proteins from purified complexes Resolves larger number of peptide fragments than traditional LC-MS/MS systems by combining strong cation exchange (SCX) and reverse phase (RP) columns (adapted from McDonald et al., 2002) Peptides identified by electrospray ionization (ESI) mass spectrometry

6. Yeast Two-Hybrid Screens Gal4 Binding DomainORF6000 genes Gal4 Activation Domain Expression of reporter gene indicates interaction between the two gene products DNA Gal4 BD ORF 6000 genes Gal4 AD Reporter Gene

7. Fluorescence Localization Provides information on where unknown proteins function in the cell ORFs were tagged with “ Venus ” yellow fluorescent protein YFP-fusions must maintain cell viability ORFVenus

8. Protein Structure Prediction Ginzu, an iterative process, was used for domain parsing and structure prediction PSI-BLAST identified remote homologies and fold recognition servers identified further matches ROSETTA was used to predict structures of unmatched domains and these were compared to known structures Combinations of these data were used to increase confidence of structure predictions

9. Gene Ontology Consortium GO is a collaborative effort to develop consistent descriptions of gene products among databases Three vocabularies are under construction to describe ORFs in terms of: 1) biological process 2) cellular localization 3) molecular/biochemical function Availability of standardized terms should simplify part of the research process www.geneontology.org

10. YDR288w & YML023c: DNA repair complexes Both complexes copurify with Smc5-Rhc18 YDR288w purified with Nse1, Mms21, and Qri2; confirmed by two hybrid screen NSE1, SMC5, and MMS21 mutants show sensitivity to DNA damaging agents YDR288w localized to nucleus (Hazbun et al., 2003)

11. YDR288w & YML023c: DNA repair complexes YML023c purifies with Kre29; confirmation by two hybrid screen YML023c also localized to nucleus YML023c purifies with other DNA repair-related proteins such as Smt3 and Pac1 (Hazbun et al., 2003)

12. YKR079c: DNA/RNA catabolism YKR079c purifies with YMR099c and Nuc1 It localized in two distinct regions: in the nucleus with YMR099c and in the mitochondria with Nuc1 Nuc1 plays a role in DNA/RNA catabolism and exhibits both ribonuclease and deoxyribonuclease activities YKR079c structure predictions supports its role in nucleic acid catabolism (Hazbun et al., 2003)

13. YLR424w, YKR022c,YLR132c: novel splicing proteins YLR424w and YKR022c purify with 18 spliceosome components They also show interaction with each other through two-hybrid screens Both localized to the nucleus YLR424w is predicted to have a G-patch domain, which is involved in RNA binding YLR132c appears to be bifunctional, playing roles in mRNA splicing and aerobic respiration

14. DNA Repair Networks This suite of protein technologies is a powerful tool for identification of functions of unknown proteins and prediction of certain networks of interactions Strong evidence that the uncharacterized proteins YDR288w and YML023c are components of two related DNA repair complexes (Hazbun et al., 2003)

15. Summary of Screening 77 ORFs annotated with at least 1 GO term 48 ORFs annotated with at least 2 GO terms 17 ORFs annotated with all 3 GO terms Fluorescence localization assigned 63 cellular component terms TAP/MudPIT assigned 32 biological process terms Two-hybrid screen assigned 16 process terms Protein structure prediction and homology searches assigned molecular function terms for 27 ORFs

16. Conclusions Integration of technologies directly assays a selected group of proteins for function Previous studies that do not combine technologies identified some, but not all, of these interactions Integrated approaches can yield large amounts of information for uncharacterized groups of proteins Modification of current protocols, or addition of new approaches should accurately predict the roles of many more uncharacterized ORFs

17. References Ashburner, M, Ball, CA, Blake, JA, Botstein, D, Butler, H, Cherry, JM, Davis, AP, Dolinski, K, Dwight, SS, Eppig, JT, Harris, MA, Hill, DP, Issel-Tarver, L, Kasarskis, A, Lewis, S, Matese, JC, Richarson, JE, Ringwald, M, and Sherlock, G. (2000) Nat. Genet. 25, 25-29. Hazbun, TR, Malmstr ö m, L, Anderson, S, Graczyk, G, Fox, B, Riffle, M, Sundin, BA, Aranda, JD, McDonald, WH, Chiu, C-H, Snydsman, BE, Bradley, P, Muller, EGD, Fields, S, Baker, D, Yates III, JR, and Davis, TN. (2003) Mol. Cell 12, 1353-1365. McDonald, WH, Ohi, R, Miyamoto, DT, Mitchison, TJ, and Yates III, JR. (2002) Int. J. Mass Spectrom. 219, 245-251. Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, and Seraphin B. (1999) Nature Biotechnol. 17, 1030-1032.