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. 2012 Dec;58(4):392-9.
doi: 10.1016/j.ymeth.2012.07.015. Epub 2012 Jul 24.

Studying protein complexes by the yeast two-hybrid system

Seesandra V Rajagopala  1 , Patricia Sikorski, J Harry Caufield, Andrey Tovchigrechko, Peter Uetz
Affiliations

Studying protein complexes by the yeast two-hybrid system

Seesandra V Rajagopala et al. Methods. 2012 Dec.

Abstract

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

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Figures

Fig. 1
Fig. 1. Mapping interactions within E. coli protein complexes by Y2H
(A) The Escherichia coli DNA polymerase III complex as determined by AP/MS (complex 42 in [7]). (B) Yeast two-hybrid matrix screening of the DNA polymerase III complex with N and C- terminal fusion baits with N-terminal fusion prey clones. Growth of yeast indicates positive PPI; auto-activators (false positive PPI) are marked as red. (C) The topology of the protein complex based on protein-protein interactions obtained in (B) as well as known direct interactions and structural information collected from the literature [–58] and PDB. (D) Protein complex 34 from [7]. No published information is available on the topology of this complex. As in panels (A–C) Y2H protein-protein interaction data was used to map the topology of this complex.
Fig. 2
Fig. 2. The ribonucleotide reductase (RNR) complex of Varicella Zoster virus (VZV)
(A) Model of the VZV RNR complex based on crystal structures of Salmonella typhimurium RNR (2BQ1 [32]). VZV ORF18 = R2 (Uniprot P09247), ORF19 = R1 (P09248) with chains in each homo-dimer numbered as I and II. R1 chain II and R2 chain II are colored in rainbow from N-terminal (blue) to C-terminal (red), with terminals labeled as NT and CT, respectively. The C-terminal of the R2 (II) chain was replaced with the corresponding peptide taken from 2BQ1 (thick red tube on the picture) where it is in contact with the groove in the R1 (I). The remaining part of R2 (II) CT is hidden because it is disordered in 2BQ1. The C-terminal of R1 (I) and the N-terminal of R2 (II) are shown as thick tubes in regions disordered in 2BQ1. The remaining terminals are behind the structures of the symmetric homo-dimers in this view and thus not labeled. (B) Interactions among RNR subunits detected in Y2H assays. The Gal4 DNA-binding (DBD) and activation domains (AD) are shown as green boxes that are either fused to the N or C-terminus of the RNR proteins. ORF18 was tested as full-length protein and as N- or C-terminal fragments (and each fragment was tested as N- and C-terminal fusion to DBD and AD). Y2H data in (B) from [16].
Figure 3
Figure 3. The proteasome
(A) Structure of the 26S proteasome from Schizosaccharomyces pombe as determined by cryo-EM density map (adapted from [41]). The core particle is shown in red, the AAA-ATPase hexamer in blue and the Rpn subunits in gold. (B) Interactions among proteasome subunits as determined by Y2H and cross-linking assays (“X-link”). Four interactions between 19S proteins and beta subunit proteins are omitted for clarity (α3-Rpt2, α4-Rpt2, α6-Rpt5, α7-Rpt5; see Supplementary Table 1). Y2H results are derived from 3 independent studies on the proteasomes of three different species (Sc = yeast [34], Ce = C. elegans [36], Hs = human [37]), crosslinking has been carried out in two species (Sc = Saccharomyces cerevisiae, Sp = Schizosaccharomyces pombe) [38, 41]. Structural and modeling results are derived from cryo-EM mapping, X-ray crystallography, and molecular modeling and used as “gold-standard” interactions, shown as grey bars [41, 60]. (C) Number of interactions in Y2H and cross-linking studies and the fraction that is seen in a structural model (red) that serves as “gold standard”. Numbers in red areas are the fraction of PPIs found in the “gold-standard” structure. (A) reproduced by permission from PNAS.
Figure 4
Figure 4. Bacteriophage lambda
(A) Phage particle and its protein components. (B) Interaction map of the proteins shown in (A). Proteins inside box are virion proteins, proteins outside the box are assembly factors that are not part of the assembled virion. It remains unclear if gpM (“M”) and gpL (“L”) are part of the virion. (C) Contribution of each vector pair to the known lambda interactome [43]. The left panel shows the number of interactions generated by each vector pair, including previously known interactions (shown in blue). The right panel illustrates the fraction of previously known interactions relative to the total number of PPIs generated by each vector. The pDEST vectors produce a high fraction of such “gold-standard” PPIs although their total number of known PPIs is still smaller than those generated by the pGBKT7/pGADC pair. Note that these numbers include 4 PPIs among regulatory proteins not in the virion. (A) and (B) after [43].
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References

    1. Jenni S, Leibundgut M, Boehringer D, Frick C, Mikolasek B, Ban N. Science. 2007;316:254–261. - PubMed
    1. Titz B, Rajagopala SV, Goll J, Hauser R, McKevitt MT, Palzkill T, Uetz P. PLoS ONE. 2008;3:e2292. - PMC - PubMed
    1. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi-Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM. Nature. 2000;403:623–627. - PubMed
    1. Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, Sahalie J, Hirozane-Kishikawa T, Gebreab F, Li N, Simonis N, Hao T, Rual JF, Dricot A, Vazquez A, Murray RR, Simon C, Tardivo L, Tam S, Svrzikapa N, Fan C, de Smet AS, Motyl A, Hudson ME, Park J, Xin X, Cusick ME, Moore T, Boone C, Snyder M, Roth FP, Barabasi AL, Tavernier J, Hill DE, Vidal M. Science. 2008;322:104–110. - PMC - PubMed
    1. Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A. Nature. 2005;433:531–537. - PubMed

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