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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Goffeau, A. et al. Life with 6000 genes. Science 274, 546–567 (1996).
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).
Fields, S. & Song, O. A novel genetic system to detect protein–protein interactions. Nature 340, 245– 246 (1989).
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).
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).
Flores, A. et al. A protein–protein interaction map of yeast RNA polymerase III. Proc. Natl Acad. Sci. USA 96, 7815– 7820 (1999).
Martzen, M. R. et al. A biochemical genomics approach for identifying genes by the activity of their products. Science 286, 1153–1155 (1999).
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).
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).
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).
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).
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).
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).
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).
Funakoshi, T., Matsuura, A., Noda, T. & Ohsumi, Y. Analyses of APG13 gene involved in autophagy in yeast, Saccharomyces cerevisiae. Gene 192, 207–213 ( 1997).
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).
Kramer, A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367– 409 (1996).
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).
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).
Nasmyth, K. Control of the yeast cell cycle by the Cdc28 protein kinase. Curr. Opin. Cell Biol. 5, 166–179 (1993).
Neubauer, G. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nature Genet. 20, 46–50 (1998).
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).
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.]
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).
Usui, T. et al. Complex formation and functional versatility of Mre11 of budding yeast in recombination. Cell 95, 705– 716 (1998).
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).
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).
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).
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).
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).
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).
Ma, H., Kunes, S., Schatz, P. J. & Botstein, D. Plasmid construction by homologous recombination in yeast. Gene 58, 201–216 (1987).
Ito, H., Fukuda, Y., Murata, K. & Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983).
Sherman, F., Fink, G. R. & Hicks, J. B. Methods in Yeast Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1986).
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).
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).
Hwang, L. H. et al. Budding yeast Cdc20: a target of the spindle checkpoint. Science 279, 1041–1044 ( 1998).
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).
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).
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).
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).
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.
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
Trends in Plant Science (2021)
Nature Communications (2021)
Classification and prediction of protein–protein interaction interface using machine learning algorithm
Scientific Reports (2021)
Variation in Pleiotropic Hub Gene Expression Is Associated with Interspecific Differences in Head Shape and Eye Size in Drosophila
Molecular Biology and Evolution (2021)