A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae

Abstract

Two large-scale yeast two-hybrid screens were undertaken to identify protein–protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The two-hybrid assay carried out by screening a protein array.
Figure 2: Data analysis software.
Figure 3: Expanded pathways shown using the software as described in the text.

References

  1. 1

    Goffeau, A. et al. Life with 6000 genes. Science 274, 546–567 (1996).

    ADS  CAS  Article  Google Scholar 

  2. 2

    Mewes, H. W., Albermann, K., Heumann, K., Liebl, S. & Pfeiffer, F. MIPS: a database for protein sequences, homology data and yeast genome information. Nucleic Acids Res. 25 , 28–30 (1997).

    CAS  Article  Google Scholar 

  3. 3

    Fields, S. & Song, O. A novel genetic system to detect protein–protein interactions. Nature 340, 245– 246 (1989).

    ADS  CAS  Article  Google Scholar 

  4. 4

    Bartel, P. L., Roecklein, J. A., SenGupta, D. & Fields, S. A protein linkage map of Escherichia coli bacteriophage T7. Nature Genet. 12, 72–77 (1996).

    CAS  Article  Google Scholar 

  5. 5

    Fromont-Racine, M., Rain, J. C. & Legrain, P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nature Genet. 16, 277–282 (1997).

    CAS  Article  Google Scholar 

  6. 6

    Flores, A. et al. A protein–protein interaction map of yeast RNA polymerase III. Proc. Natl Acad. Sci. USA 96, 7815– 7820 (1999).

    ADS  CAS  Article  Google Scholar 

  7. 7

    Martzen, M. R. et al. A biochemical genomics approach for identifying genes by the activity of their products. Science 286, 1153–1155 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Hudson, J. R. Jr et al. The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genome Res. 7, 1169–1173 (1997).

    CAS  Article  Google Scholar 

  9. 9

    James, P., Halladay, J. & Craig, E. A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144 , 1425–1436 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Hodges, P. E., McKee, A. H., Davis, B. P., Payne, W. E. & Garrels, J. I. The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data. Nucleic Acids Res. 27, 69–73 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Legrain, P., Dokhelar, M. C. & Transy, C. Detection of protein–protein interactions using different vectors in the two-hybrid system. Nucleic Acids Res. 22, 3241–3242 ( 1994).

    CAS  Article  Google Scholar 

  12. 12

    Ramesh, V., Gusella, J. F. & Shih, V. E. Molecular pathology of gyrate atrophy of the choroid and retina due to ornithine aminotransferase deficiency. Mol. Biol. Med. 8, 81–93 ( 1991).

    CAS  PubMed  Google Scholar 

  13. 13

    Scott, S. V. & Klionsky, D. J. Delivery of proteins and organelles to the vacuole from the cytoplasm. Curr. Opin. Cell Biol. 10, 523–529 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Scott, S. V. et al. Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole. Proc. Natl Acad. Sci. USA 93, 12304–12308 (1996).

    ADS  CAS  Article  Google Scholar 

  15. 15

    Funakoshi, T., Matsuura, A., Noda, T. & Ohsumi, Y. Analyses of APG13 gene involved in autophagy in yeast, Saccharomyces cerevisiae. Gene 192, 207–213 ( 1997).

    CAS  Article  Google Scholar 

  16. 16

    Kim, J., Scott, S. V., Oda, M. N. & Klionsky, D. J. Transport of a large oligomeric protein by the cytoplasm to vacuole protein targeting pathway. J. Cell Biol. 137, 609– 618 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Kramer, A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367– 409 (1996).

    CAS  Article  Google Scholar 

  18. 18

    Mayes, A. E., Verdone, L., Legrain, P. & Beggs, J. D. Characterization of Sm-like proteins in yeast and their association with U6 snRNA. EMBO J. 18, 4321–4331 ( 1999).

    CAS  Article  Google Scholar 

  19. 19

    Kambach, C. et al. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96 , 375–387 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Nasmyth, K. Control of the yeast cell cycle by the Cdc28 protein kinase. Curr. Opin. Cell Biol. 5, 166–179 (1993).

    CAS  Article  Google Scholar 

  21. 21

    Neubauer, G. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nature Genet. 20, 46–50 (1998).

    CAS  Article  Google Scholar 

  22. 22

    Boeck, R., Lapeyre, B., Brown, C. E. & Sachs, A. B. Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant. Mol. Cell. Biol. 18, 5062–5072 ( 1998).

    CAS  Article  Google Scholar 

  23. 23

    Kadowaki, T. et al. Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants. J. Cell Biol. 126, 649–659 (1994). [Published erratum appears in J. Cell Biol. 126, 1627.]

    CAS  Article  Google Scholar 

  24. 24

    Hollingsworth, N. M., Ponte, L. & Halsey, C. MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 9, 1728–1739 (1995).

    CAS  Article  Google Scholar 

  25. 25

    Usui, T. et al. Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95, 705– 716 (1998).

    CAS  Article  Google Scholar 

  26. 26

    Bishop, D. K., Park, D., Xu, L. & Kleckner, N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69, 439–456 (1992).

    CAS  Article  Google Scholar 

  27. 27

    SenGupta, D. J. et al. A three-hybrid system to detect RNA–protein interactions in vivo. Proc. Natl Acad. Sci. USA 93, 8496–8501 (1996).

    ADS  CAS  Article  Google Scholar 

  28. 28

    Wang, M. M. & Reed, R. R. Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364, 121–126 ( 1993).

    ADS  CAS  Article  Google Scholar 

  29. 29

    Li, J. J. & Herskowitz, I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system [see comments]. Science 262, 1870–1874 (1993).

    ADS  CAS  Article  Google Scholar 

  30. 30

    Licitra, E. J. & Liu, J. O. A three-hybrid system for detecting small ligand–protein receptor interactions. Proc. Natl Acad. Sci. USA 93, 12817–12821 (1996).

    ADS  CAS  Article  Google Scholar 

  31. 31

    Belshaw, P. J., Ho, S. N., Crabtree, G. R. & Schreiber, S. L. Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc. Natl Acad. Sci. USA 93, 4604–4607 ( 1996).

    ADS  CAS  Article  Google Scholar 

  32. 32

    Ma, H., Kunes, S., Schatz, P. J. & Botstein, D. Plasmid construction by homologous recombination in yeast. Gene 58, 201–216 (1987).

    CAS  Article  Google Scholar 

  33. 33

    Ito, H., Fukuda, Y., Murata, K. & Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Sherman, F., Fink, G. R. & Hicks, J. B. Methods in Yeast Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1986).

    Google Scholar 

  35. 35

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  Article  Google Scholar 

  36. 36

    Minvielle-Sebastia, L., Preker, P. J., Wiederkehr, T., Strahm, Y. & Keller, W. The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3′-end formation. Proc. Natl Acad. Sci. USA 94, 7897–7902 (1997).

    ADS  CAS  Article  Google Scholar 

  37. 37

    Hwang, L. H. et al. Budding yeast Cdc20: a target of the spindle checkpoint. Science 279, 1041–1044 ( 1998).

    ADS  CAS  Article  Google Scholar 

  38. 38

    Guenette, S., Magendantz, M. & Solomon, F. Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes. J. Cell Sci. 108, 1195–1204 (1995).

    CAS  PubMed  Google Scholar 

  39. 39

    Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66 , 507–517 (1991).

    CAS  Article  Google Scholar 

  40. 40

    Seeley, T. W., Wang, L. & Zhen, J. Y. Phosphorylation of human MAD1 by the BUB1 kinase in vitro. Biochem. Biophys. Res. Commun. 257, 589–595 (1999).

    CAS  Article  Google Scholar 

  41. 41

    Kallio, M., Weinstein, J., Daum, J. R., Burke, D. J. & Gorbsky, G. J. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J. Cell Biol. 141, 1393–1406 (1998).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank K. Furtak, J. Gilbert, N. Huber, M. Laurino, L. Matthies, A. Perna, C. Pratt and B. Rittman for technical assistance; B. Drees, R. Hughes and S. McCraith for help with some of the experiments; P. Hodges (Proteome) for providing a compilation of protein interactions; and B. Byers, M. Olson, R. Franza, M. Roth, D. Lewin, T. Jarvie and J. Simons for comments on the manuscript. S.F. is supported by grants from the NIH and the Merck Genome Research Institute. P.U. is supported by a fellowship from the Deutscher Akademischer Austauschdienst (DAAD). S.F. is an investigator of the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Stanley Fields.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Uetz, P., Giot, L., Cagney, G. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000). https://doi.org/10.1038/35001009

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing