Using λ repressor fusions we were able to identify potential homotypic interactions in 35 proteins encoded by the yeast genome, including one protein from the 2μ plasmid present in the strain used to make the libraries. About half of the ISTs represent previously identified interactions, while the rest have not been described before. The ISTs we identify also represent a combination of proteins of known structure, those for which structures can be predicted with reasonably high confidence, and proteins of unknown structure.
In principle, all of the identities of yeast proteins capable of self-assembly should show up in an all vs. all interaction screen, such as the large-scale two-hybrid studies being undertaken by several laboratories. What, then, is the benefit of using the repressor fusion approach? shows a Venn diagram representation of the homotypic interactions found in this study and in three different two-hybrid studies: two using full-length ORFs from Ito et al. (2001)
and Uetz et al. (2000)
and one using predicted coiled-coil domains from Newman et al. (2000)
. There is minimal overlap between our results and the three large-scale yeast two-hybrid studies. The overlap among the yeast two-hybrid datasets is similarly small. Thus, most of the homotypic interactions we have found were not found in the earlier studies.
As noted by Hazbun and Fields (2001)
, despite the efforts to make each of the studies comprehensive, many interactions known from biochemical data have not been found by any of the large-scale interaction screens. In the study reported here we are clearly far from saturation. Interactions known to be detectable in reconstruction experiments, most notably the Gcn4p leucine zipper, have not yet been found. Although it is likely that additional screening of existing libraries will yield new ISTs, our libraries are also likely to be biased by the non-random cleavage of Cvi
TI sites in our partial digests. New libraries based on other ways to fragment the target DNA may be a richer source of new ISTs.
In practice, comprehensive identification of protein-protein interactions will involve complementary information from a variety of genetic and biochemical approaches. Among the genetic approaches, repressor fusions are well suited to identify homotypic interactions. Newman et al. (2000)
has argued that homotypic interactions, especially those involving homodimers are likely to be underrepresented in yeast two-hybrid screens due to preferential interaction of baits within a dimeric DNA binding protein over preys coming from solution. A wide variety of technical limitations will affect the recovery of ISTs from yeast two-hybrid pairs, repressor fusions or both, e.g. post-translational modifications required for folding in assembly in yeast are unlikely to be recapitulated in E. coli
. Nevertheless, both two-hybrid methods and repressor fusions can clearly provide identities of many proteins involved in homotypic interactions.
A genome-wide survey of protein-protein interactions should provide two kinds of information: not only what proteins can interact, but also what parts of the proteins are involved in the interactions. One of the most useful kinds of information provided by repressor fusions is the localization of oligomerization domains on a genome-wide scale. Because repressor fusions require only single libraries of hybrid proteins to identify homotypic ISTs, the number of subdomains that can be tested scales linearly with the number of clones that can be subjected to selection for repressor activity. By contrast, detecting a homotypic interaction in a two-hybrid system requires that both the bait and prey be present in the same cell. This means that the number of protein fragments that can be tested scales only as the square root of the library size.
These considerations, along with the higher transformation efficiency of E. coli
, allowed us to use random fragments of genomic DNA instead of the full-length ORFs favored by the large-scale yeast two-hybrid approaches. Thus, our ISTs provide mapping information about the location of the oligomerization domains within proteins as well as the identities of the proteins involved in self-assembly. In general, the ISTs we find are much smaller than the proteins that contain them. Where different, overlapping ISTs are recovered from the same protein (as for Hsp26p, Mdj1p, Not5p, Skn7p, Srl2p, Tup1p and Yap5p) the endpoints of the ISTs can be used to delimit the minimal region required for oligomerization. In the case of Tup1p, amino acids 1-72 have been shown to be sufficient for oligomerization (Tzamarias and Struhl, 1994
; Varanasi et al., 1996
). The shortest IST we found covered amino acids 1-119, while the overlap between two ISTs suggested that residues 12-119 might be sufficient to form an oligomer.
Self-assembling domains derived from IST analysis will expand our understanding of the many ways nature builds protein complexes. The domains may provide more tractable targets for structure determination than the intact proteins from which they come. Additionally, isolated interaction domains may provide useful tools for functional genomics; expression of the domains in yeast could yield dominant negative phenotypes. While this may not provide much new information in a genetically well-characterized system like S. cerevisiae, a similar approach may be useful for a variety of genetically less tractable organisms. In cases where assembly domains prove to be involved in an important cellular function, the repressor fusions themselves can provide screens for drug discovery.
Finally, detailed study of homotypic interaction domains is likely to identify new structural motifs that will be found in other proteins. Although the interactions we identify are homotypic, it is likely that in many cases, evolutionarily related structures are also used for heterotypic interactions. Examples of structures used in both homotypic and heterotypic interactions include the HLH (Robinson and Lopes, 2000
) and leucine zipper (Hurst, 1994
) motifs. Other interaction domains that function in both homo- and hetero-oligomers may provide additional mechanisms of regulation by combinatorial assembly of different subunits.