- Email: [email protected]
A cytoplasmic protein–protein interaction detection method based on reporter translation
Analytical Biochemistry 384 (2009) 362–364
Contents lists available at ScienceDirect
Analytical Biochemistry j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b i o
Notes & Tips
A cytoplasmic protein–protein interaction detection method based on reporter translation Laurence Renaut *, Khalil Bouayadi, Hakim Kharrat, Philippe Mondon MilleGen, Immeuble BIOSTEP, 31681 Labege Cedex, France
a r t i c l e
i n f o
Article history: Received 31 July 2008 Available online 17 October 2008
a b s t r a c t One approach to drug discovery involves the targeting of abnormal protein–protein interactions that lead to pathology. We present a new technology allowing the detection of such interactions within the cytoplasm in a yeast-based system. The interaction detection is based on the sequestration of a trans lation termination factor involved in stop codon recognition. This sequestration inhibits the activity of the factor, thereby permitting the translation of a reporter gene harboring a premature stop codon. This novel cytoplasmic protein–protein interaction (CPPI) detection system should prove to be useful in the characterization of proteins as well as in partner identification, interaction mapping, and drug discovery applications. © 2008 Elsevier Inc. All rights reserved.
The human genome consists of 20,000–30,000 genes coding for more than 500,000 different proteins. Rather than operating alone, more than 80% of these proteins form complexes. Protein–protein interaction mechanisms are involved in many cellular events and pathol ogical development. As such, a fuller understanding of these mechanisms could help in the discovery of new therap eutic targets and in the conception of new drugs. Many tools are currently avail able to study such interactions, but all have their drawbacks, mainly involving a reduced compatibility between the detection system and the properties of the protein (e.g., function, cellular localiza tion, posttranslational modification) [1,2]. Hence, new technolo gies are needed to develop a variety of methods that would allow the study of a wide range of proteins involved in protein–protein interactions. With this in mind, and with regard to protein diver sity, we developed a new technology allowing the direct analysis of cytoplasmic protein–protein interactions (CPPIs).1 As with mammalian prion proteins, the yeast prion proteins are present in either aggregated or soluble form; the aggregated form is able to convert the soluble into an aggregated form [3]. Sup35 is a yeast prion protein composed of three domains. The Sup35 N-terminal domain and M domain (Sup35NM) are respon sible for Sup35 aggregation and maintenance of the aggregated form [4]. The Sup35 C-terminal domain (Sup35C) has been shown to contain a stop codon recognition domain. The sequestration of Sup35C into aggregates within the cytoplasm inhibits the stop translation activity [5]. We used a yeast [PSI+] strain containing * Corresponding author. Fax: +33 5 61 28 70 11. E-mail address: laurence.renaut@millegen.com (L. Renaut). 1 Abbreviations used: CPPI, cytoplasmic protein–protein interaction; Sup35NM, Sup35 N-terminal domain and M domain; Sup35C, Sup35 C-terminal domain; WT, wild-type; Y2H, yeast two-hybrid; PCA, protein complementation assay. 0003-2697/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.10.011
endogenous aggregated Sup35 prion protein. In such a strain, the read-through of premature stop codons has been reported in some genes when Sup35 is aggregated, thereby resulting in a particular phenotype [6]. Using the characteristics of Sup35NM and Sup35C, we developed an interaction detection system based on the lacZ reporter gene harboring a premature stop codon. This system was developed with a pair of well-characterized interacting proteins: P53 as bait and miniT as prey; the miniT corresponds to the SV40 T antigen domain containing the P53 binding site [7,8]. A lacZ reporter gene mutant (W13) harboring a premature stop codon (TGG548 ! TGA) was selected among 17 mutants for its dependence on a Sup35 aggregation state for translation (Fig. 1B). As depicted in Fig. 1A, the endogenous Sup35 in the yeast [PSI+] strain is aggregated, thereby leading to a read-through of the reporter lacZ W13 mutant. The lacZ W13 translation depen dence on Sup35 aggregation was tested using GuHCl, a compound known to solubilize Sup35 aggregates. Results in Fig. 1B show that, contrary to wild-type (WT) lacZ, the lacZ W13 mutant translation is dependent on Sup35 aggregation. The requirement of the detection system resides in the fusion activity of Sup35C. In theory, the fusion of Sup35C to a P53 protein should change the phenotype associated with the lacZ W13 reporter gene. Indeed, the Sup35C part of the fusion protein should stop the translation of the reporter gene at the premature stop codon (Fig. 1C). No data exist, however, regarding the ability of Sup35C fused to heterologous protein to stop translation simi larly to the full-length Sup35 protein. Therefore, we cotransformed the yeast 74D694 [PSI+] strain with the W13 lacZ mutant gene and the P53–Sup35C fusion protein or an empty vector (control). We then monitored the capacity of the P53–Sup35C fusion protein to decrease the W13 mutant translation of five isolated colonies
Notes & Tips / Anal. Biochem. 384 (2009) 362–364
363
Endogen sup 35 aggregates
Endogen sup35 aggregates P53-sup35C fusion
ATG
sup35NM aggregation domain
polyA
ATG
polyA
sup35C stop codon recognition factor P53 β-galactosidase
relative π-galactosidase activity
1
Percentage of β-galactosidase activity
100
50
0 0
3
GuHCL concentration [µM]
0.5
0 W13
W13+P53Sup35C
Fig. 1. Effect of target protein fused to Sup35C on yeast phenotype. (A) Schematic representation of W13 lacZ mutant gene expression in the [PSI+] strain. (B) Test of the effect of Sup35 aggregation status on the translation of W13 lacZ mutant (gray) and WT lacZ (black). Endogen, endogenous. (C) Schematic representation of P53–Sup35C fusion expression effect on W13 translation. (D) Measure of the lacZ W13 mutant activity with P53–Sup35C fusion.
from each condition (Fig. 1D). The results showed that when fused to P53, Sup35C was still able to recognize the lacZ W13 stop premature codon and inhibit lacZ W13 expression. This control was essential to ensure the interaction with a given bait protein. Fused to bait protein, Sup35C activity can be easily observed, as a phenotypic change and the feasibility of the study confirmed. In the case of nonfunctionality of the fusion protein, a design of the bait protein can be performed. This design will ensure the compat ibility between Sup35C and the bait protein in a fusion context and enable its use in our system. The ability of Sup35NM fused to a heterologous protein to form aggregates has been well described and has included proteins harboring different proprieties: the Gag-P55 antigen of HIV-1, green fluorescent protein, and a transcription factor (glucocorti coid receptor) [9–11]. We concluded that a heterologous protein fused to Sup35NM should be found in aggregates. The principle behind this interacting system, illustrated with P53 and miniT, is described in Fig. 2. An R3 peptide that does not bind P53 was used as a negat ive control. The fusion protein comprising miniT or R3 fused to Sup35NM was coexpressed with P53–Sup35C fusion protein and lacZ W13 mutant. As P53 and miniT interacted, we expected colonies containing Sup35NM–miniT and P53–Sup35C fusions to show a phenotype associated with a read-through of the W13 lacZ premature stop codon due to Sup35C sequestration within aggregates via miniT–P53 interaction (Fig. 2A). In contrast, we expected the negative control corresponding to Sup35NM–R3 and P53–Sup35C to generate a phenotype associated with the incomplete translation of the reporter gene. Because P53 and R3 did not interact, the P53–Sup35C fusion remained free to stop translation of the lacZ W13 reporter protein (Fig. 2B). Because overexpression of Sup35NM has been described as being harmful for yeast viability [12], the expression of the Sup35NM–miniT fusion protein was placed under the control of the PCUP-1 promoter (CuSO4 inducible). We tested the capacity of the Sup35NM–miniT fusion to restore the W13 lacZ expres sion by cotransforming the yeast with W13 lacZ, P53–Sup35C, and either the interacting fusion Sup35NM–miniT or the noninteract
ing fusion Sup35NM–R3. Increasing concentrations of CuSO4 were used to induce the Sup35NM fusion expression and the W13 lacZ mutant expression measured (Fig. 2C). As expected, the expression of Sup35NM–miniT fusion, but not Sup35NM–R3 fusion, restored the W13 lacZ expression. The restored expression of the W13 lacZ mutant was proportional to the level of induction of 35NM–mi niT expression by CuSO4. Results show that the coexpression of the two fusion proteins containing interacting moieties led to a significantly detectable expression of the lacZ W13 mutant. Finally, we have shown that the Sup35NM–miniT effect on the expression of lacZ W13 is dependent on the presence of P53 fused to Sup35C. Fig. 2D describes the effect of Sup35NM–miniT on lacZ W13 expression in the presence of P53–Sup35C or Sup35C alone. Expression of P53–Sup35C fusion or Sup35C alone reduced the expression of lacZ W13. However, as shown earlier, the coexpres sion of Sup35NM–miniT with P53–Sup35C fusion led to a strong expression of the W13 lacZ mutant. This phenomenon was not observed with Sup35C alone coexpressed with the Sup35NM–mi niT fusion. The data provide evidence for the effect of Sup35NM–miniT on lacZ W13 expression being specific to the presence of P53 fused to Sup35C. The interaction of miniT and P53 can be monitored in the cytoplasm of the yeast via the lacZ W13 mutant expression detection. Many cellular processes are regulated by protein–protein inter action, and our technology is an excellent tool to advance inter actome understanding. Furthermore, many cellular events such as those involved in signaling pathways occur in the cytoplasm, with some interactions requiring posttranslational modifications of the interacting partners. Because our technology allows the detec tion of protein interactions within the cytoplasm, it is a pertinent approach to studying protein interactions in their natural subcel lular compartment. Yeast-based methods, such as Y2H (yeast two-hybrid) [13] and PCA (protein complementation assay) [14], are widely used to study protein interactions. Our technology provides a good alternative to these in vivo methods when performing interaction studies for a given bait protein. Furthermore, similar to Y2H and
364
Notes & Tips / Anal. Biochem. 384 (2009) 362–364
Interaction (Sup35NM-miniT)
No interaction (Sup35NM-R3)
↔ Endogen sup 35 aggregates
Endogen sup 35 aggregates
↔ ↔
ATG
polyA
ATG
Sup35NM aggregation domain Sup35C stop codon recognition factor
CuSO4 (20µM)
P53 R3 miniT
0
W13+sup35NM-miniT
W13+P53sup35C+sup35NMminiT
W13+P53sup35C+sup35NMR3
W13+P53-sup35C
0
0.7
W13+sup35 C+sup35NM-miniT
CuSO4 (10µM)
CuSO4 (0µM) CuSO4 (5µM) CuSO4 (10µM) CuSO4 (20µM)
1.4
W13+sup35C
β-galactosidase
CuSO4 (5µM)
W13+P53sup35C+sup35NM-miniT
CuSO4 (2µM)
W13+P53-sup35C
CuSO4 (0µM)
relativeβ-galactosidaseactivity
Relativeβ-galactosidse activity
1
0.5
polyA Reporter protein
Fig. 2. Effect of presence of both partners fused to Sup35C and Sup35NM on yeast phenotype. (A,B) Schematic representations of expected results when expressing Sup35NM– miniT (A) or Sup35NM–R3 (B). Endog en, endogenous. (C) Measure of W13 expression with a pair of interacting proteins or noninteracting proteins. (D) Test of the influence of P53 fused to Sup35C on the Sup35NM–miniT effect on W13 expression restoration.
PCA, in our system bait and prey proteins are in a translational fusion format. Our technology allows us to ensure the functionality of Sup35C fused to a given bait protein before pursuing the inter action studies. Y2H has been successfully used to identify protein– protein interactions in several model organisms. Nevertheless, the interaction in Y2H occurs in the nucleus, which is not the natural cellular compartment of many proteins of interest for interaction studies. In contrast, our system provides a direct detection in the cytoplasm, where the posttranslational modifications are impor tant for protein interaction. This technology should prove to be useful in the characterization of proteins as well as in partner identification, interaction map ping, and drug discovery applications. It may also be adapted in the development of a tool for use in drug discovery such as the screen ing of small molec ules, peptides, or intrabodies able to modulate intracytoplasmic interactions in a high-throughput screening format. For this purpose, other reporter genes and expression cas settes are currently optimized for use in studies that could be the basis of developing drugs for use in disorders often involving the pathol ogical deregulation of otherwise finely regulated cellular protein interactions. Acknowledgments We thank M. Blondel (U613-INSERM, Brest) for providing the yeast 74D694 strain and yeast expression plasmids and for engaging in helpful discussions at the beginning of this project. We thank MilleGen’s sequencing service for the quality of the results. We also thank our collaborators: M.J. Carles for techni cal support and A. Deflisque, C. Monnet, O. Dubreuil, F. Crozet, and P. Brune for discussions and critical reviews of the manu script.
References [1] J. Piehler, New methodologies for measuring protein interactions in vivo and in vitro, Curr. Opin. Struct. Biol. 15 (2005) 4–14. [2] T. Berggård, S. Linse, P. James, Methods for the detection and analysis of pro tein–protein interactions, Proteomics 7 (2007) 2833–2842. [3] S.B. Prusiner, Novel proteinac eous infectious particles cause scrapie, Science 216 (1982) 136–144. [4] M.D. Ter-Avanesyan, A.R. Dagkesamanskaya, V.V. Kushnirov, V.N. Smirnov, The SUP35 omnipotent suppressor gene is involved in the maintenance of the nonMendelian determinant [PSI+] in the yeast Saccharomyces cerevisiae, Genetics 137 (1994) 671–676. [5] I. Stansfield, K.M. Jones, V.V. Kushnirov, A.R. Dagkesamanskaya, A.I. Poznya kovski, S.V. Paushkin, C.R. Nierras, B.S. Cox, M.D. Ter-Avanesyan, M.F. Tuite, The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae, EMBO J. 14 (1995) 4365–4373. [6] A.L. Manogaran, K.T. Kirkland, S.W. Liebman, An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence, and appear ance of the [PSI+] prion in Saccharomyces cerevisiae, Yeast 23 (2006) 141–147. [7] J.S. Butel, J.A. Lednicky, Cell and molecul ar biology of Simian virus 40: implica tions for human infections and disease, J. Natl. Cancer Inst. 91 (1999) 119–134. [8] E. Fanning, Simian virus 40 large T antigen: the puzzle, the pieces, and the emerging picture, J. Virol. 66 (1992) 1289–1293. [9] P.A. Ivanov, E.I. Lewitin, B.I. Shevelev, G.V. Fominov, J.A. Wojciechowska, A.H. Asadi Mobarhan, Y.V. Vertie, N.K. Yankovsky, A.B. Shevelev, Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of Botulinum neurotoxin in Saccharomyces cerevisiae, Res. Microbiol. 152 (2001) 27–35. [10] P. Zhou, I.L. Derkatch, S.W. Liebman, The relationship between visible intra cellular aggregates that appear after overexpression of Sup35 and the yeast prion-like elements [PSI(+)] and [PIN(+)], Mol. Microbiol. 39 (2001) 37–46. [11] S. Lindquist, S. Krobitsch, L. Li, N. Sondheimer, Investigating protein conforma tion-based inheritance and disease in yeast, Philos. Trans. R. Soc. Lond. B 356 (2001) 169–176. [12] E.E. Ganusova, L.N. Ozolins, S. Bhagat, G.P. Newnam, R.D. Wegrzyn, M.Y. Sher man, Y.O. Chernoff, Modulation of prion formation, aggregation, and toxicity by the actin cytoskeleton in yeast, Mol. Cell. Biol. 26 (2006) 617–629. [13] S. Fields, O. Song, A novel genetic system to detect protein–protein interac tions, Nature 340 (1989) 245–246. [14] N. Johnsson, A. Varshavsky, Split ubiquitin as a sensor of protein interactions in vivo, Proc. Natl. Acad. Sci. USA 91 (1994) 10340–10344.
Contents lists available at ScienceDirect
Analytical Biochemistry j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b i o
Notes & Tips
A cytoplasmic protein–protein interaction detection method based on reporter translation Laurence Renaut *, Khalil Bouayadi, Hakim Kharrat, Philippe Mondon MilleGen, Immeuble BIOSTEP, 31681 Labege Cedex, France
a r t i c l e
i n f o
Article history: Received 31 July 2008 Available online 17 October 2008
a b s t r a c t One approach to drug discovery involves the targeting of abnormal protein–protein interactions that lead to pathology. We present a new technology allowing the detection of such interactions within the cytoplasm in a yeast-based system. The interaction detection is based on the sequestration of a trans lation termination factor involved in stop codon recognition. This sequestration inhibits the activity of the factor, thereby permitting the translation of a reporter gene harboring a premature stop codon. This novel cytoplasmic protein–protein interaction (CPPI) detection system should prove to be useful in the characterization of proteins as well as in partner identification, interaction mapping, and drug discovery applications. © 2008 Elsevier Inc. All rights reserved.
The human genome consists of 20,000–30,000 genes coding for more than 500,000 different proteins. Rather than operating alone, more than 80% of these proteins form complexes. Protein–protein interaction mechanisms are involved in many cellular events and pathol ogical development. As such, a fuller understanding of these mechanisms could help in the discovery of new therap eutic targets and in the conception of new drugs. Many tools are currently avail able to study such interactions, but all have their drawbacks, mainly involving a reduced compatibility between the detection system and the properties of the protein (e.g., function, cellular localiza tion, posttranslational modification) [1,2]. Hence, new technolo gies are needed to develop a variety of methods that would allow the study of a wide range of proteins involved in protein–protein interactions. With this in mind, and with regard to protein diver sity, we developed a new technology allowing the direct analysis of cytoplasmic protein–protein interactions (CPPIs).1 As with mammalian prion proteins, the yeast prion proteins are present in either aggregated or soluble form; the aggregated form is able to convert the soluble into an aggregated form [3]. Sup35 is a yeast prion protein composed of three domains. The Sup35 N-terminal domain and M domain (Sup35NM) are respon sible for Sup35 aggregation and maintenance of the aggregated form [4]. The Sup35 C-terminal domain (Sup35C) has been shown to contain a stop codon recognition domain. The sequestration of Sup35C into aggregates within the cytoplasm inhibits the stop translation activity [5]. We used a yeast [PSI+] strain containing * Corresponding author. Fax: +33 5 61 28 70 11. E-mail address: laurence.renaut@millegen.com (L. Renaut). 1 Abbreviations used: CPPI, cytoplasmic protein–protein interaction; Sup35NM, Sup35 N-terminal domain and M domain; Sup35C, Sup35 C-terminal domain; WT, wild-type; Y2H, yeast two-hybrid; PCA, protein complementation assay. 0003-2697/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.10.011
endogenous aggregated Sup35 prion protein. In such a strain, the read-through of premature stop codons has been reported in some genes when Sup35 is aggregated, thereby resulting in a particular phenotype [6]. Using the characteristics of Sup35NM and Sup35C, we developed an interaction detection system based on the lacZ reporter gene harboring a premature stop codon. This system was developed with a pair of well-characterized interacting proteins: P53 as bait and miniT as prey; the miniT corresponds to the SV40 T antigen domain containing the P53 binding site [7,8]. A lacZ reporter gene mutant (W13) harboring a premature stop codon (TGG548 ! TGA) was selected among 17 mutants for its dependence on a Sup35 aggregation state for translation (Fig. 1B). As depicted in Fig. 1A, the endogenous Sup35 in the yeast [PSI+] strain is aggregated, thereby leading to a read-through of the reporter lacZ W13 mutant. The lacZ W13 translation depen dence on Sup35 aggregation was tested using GuHCl, a compound known to solubilize Sup35 aggregates. Results in Fig. 1B show that, contrary to wild-type (WT) lacZ, the lacZ W13 mutant translation is dependent on Sup35 aggregation. The requirement of the detection system resides in the fusion activity of Sup35C. In theory, the fusion of Sup35C to a P53 protein should change the phenotype associated with the lacZ W13 reporter gene. Indeed, the Sup35C part of the fusion protein should stop the translation of the reporter gene at the premature stop codon (Fig. 1C). No data exist, however, regarding the ability of Sup35C fused to heterologous protein to stop translation simi larly to the full-length Sup35 protein. Therefore, we cotransformed the yeast 74D694 [PSI+] strain with the W13 lacZ mutant gene and the P53–Sup35C fusion protein or an empty vector (control). We then monitored the capacity of the P53–Sup35C fusion protein to decrease the W13 mutant translation of five isolated colonies
Notes & Tips / Anal. Biochem. 384 (2009) 362–364
363
Endogen sup 35 aggregates
Endogen sup35 aggregates P53-sup35C fusion
ATG
sup35NM aggregation domain
polyA
ATG
polyA
sup35C stop codon recognition factor P53 β-galactosidase
relative π-galactosidase activity
1
Percentage of β-galactosidase activity
100
50
0 0
3
GuHCL concentration [µM]
0.5
0 W13
W13+P53Sup35C
Fig. 1. Effect of target protein fused to Sup35C on yeast phenotype. (A) Schematic representation of W13 lacZ mutant gene expression in the [PSI+] strain. (B) Test of the effect of Sup35 aggregation status on the translation of W13 lacZ mutant (gray) and WT lacZ (black). Endogen, endogenous. (C) Schematic representation of P53–Sup35C fusion expression effect on W13 translation. (D) Measure of the lacZ W13 mutant activity with P53–Sup35C fusion.
from each condition (Fig. 1D). The results showed that when fused to P53, Sup35C was still able to recognize the lacZ W13 stop premature codon and inhibit lacZ W13 expression. This control was essential to ensure the interaction with a given bait protein. Fused to bait protein, Sup35C activity can be easily observed, as a phenotypic change and the feasibility of the study confirmed. In the case of nonfunctionality of the fusion protein, a design of the bait protein can be performed. This design will ensure the compat ibility between Sup35C and the bait protein in a fusion context and enable its use in our system. The ability of Sup35NM fused to a heterologous protein to form aggregates has been well described and has included proteins harboring different proprieties: the Gag-P55 antigen of HIV-1, green fluorescent protein, and a transcription factor (glucocorti coid receptor) [9–11]. We concluded that a heterologous protein fused to Sup35NM should be found in aggregates. The principle behind this interacting system, illustrated with P53 and miniT, is described in Fig. 2. An R3 peptide that does not bind P53 was used as a negat ive control. The fusion protein comprising miniT or R3 fused to Sup35NM was coexpressed with P53–Sup35C fusion protein and lacZ W13 mutant. As P53 and miniT interacted, we expected colonies containing Sup35NM–miniT and P53–Sup35C fusions to show a phenotype associated with a read-through of the W13 lacZ premature stop codon due to Sup35C sequestration within aggregates via miniT–P53 interaction (Fig. 2A). In contrast, we expected the negative control corresponding to Sup35NM–R3 and P53–Sup35C to generate a phenotype associated with the incomplete translation of the reporter gene. Because P53 and R3 did not interact, the P53–Sup35C fusion remained free to stop translation of the lacZ W13 reporter protein (Fig. 2B). Because overexpression of Sup35NM has been described as being harmful for yeast viability [12], the expression of the Sup35NM–miniT fusion protein was placed under the control of the PCUP-1 promoter (CuSO4 inducible). We tested the capacity of the Sup35NM–miniT fusion to restore the W13 lacZ expres sion by cotransforming the yeast with W13 lacZ, P53–Sup35C, and either the interacting fusion Sup35NM–miniT or the noninteract
ing fusion Sup35NM–R3. Increasing concentrations of CuSO4 were used to induce the Sup35NM fusion expression and the W13 lacZ mutant expression measured (Fig. 2C). As expected, the expression of Sup35NM–miniT fusion, but not Sup35NM–R3 fusion, restored the W13 lacZ expression. The restored expression of the W13 lacZ mutant was proportional to the level of induction of 35NM–mi niT expression by CuSO4. Results show that the coexpression of the two fusion proteins containing interacting moieties led to a significantly detectable expression of the lacZ W13 mutant. Finally, we have shown that the Sup35NM–miniT effect on the expression of lacZ W13 is dependent on the presence of P53 fused to Sup35C. Fig. 2D describes the effect of Sup35NM–miniT on lacZ W13 expression in the presence of P53–Sup35C or Sup35C alone. Expression of P53–Sup35C fusion or Sup35C alone reduced the expression of lacZ W13. However, as shown earlier, the coexpres sion of Sup35NM–miniT with P53–Sup35C fusion led to a strong expression of the W13 lacZ mutant. This phenomenon was not observed with Sup35C alone coexpressed with the Sup35NM–mi niT fusion. The data provide evidence for the effect of Sup35NM–miniT on lacZ W13 expression being specific to the presence of P53 fused to Sup35C. The interaction of miniT and P53 can be monitored in the cytoplasm of the yeast via the lacZ W13 mutant expression detection. Many cellular processes are regulated by protein–protein inter action, and our technology is an excellent tool to advance inter actome understanding. Furthermore, many cellular events such as those involved in signaling pathways occur in the cytoplasm, with some interactions requiring posttranslational modifications of the interacting partners. Because our technology allows the detec tion of protein interactions within the cytoplasm, it is a pertinent approach to studying protein interactions in their natural subcel lular compartment. Yeast-based methods, such as Y2H (yeast two-hybrid) [13] and PCA (protein complementation assay) [14], are widely used to study protein interactions. Our technology provides a good alternative to these in vivo methods when performing interaction studies for a given bait protein. Furthermore, similar to Y2H and
364
Notes & Tips / Anal. Biochem. 384 (2009) 362–364
Interaction (Sup35NM-miniT)
No interaction (Sup35NM-R3)
↔ Endogen sup 35 aggregates
Endogen sup 35 aggregates
↔ ↔
ATG
polyA
ATG
Sup35NM aggregation domain Sup35C stop codon recognition factor
CuSO4 (20µM)
P53 R3 miniT
0
W13+sup35NM-miniT
W13+P53sup35C+sup35NMminiT
W13+P53sup35C+sup35NMR3
W13+P53-sup35C
0
0.7
W13+sup35 C+sup35NM-miniT
CuSO4 (10µM)
CuSO4 (0µM) CuSO4 (5µM) CuSO4 (10µM) CuSO4 (20µM)
1.4
W13+sup35C
β-galactosidase
CuSO4 (5µM)
W13+P53sup35C+sup35NM-miniT
CuSO4 (2µM)
W13+P53-sup35C
CuSO4 (0µM)
relativeβ-galactosidaseactivity
Relativeβ-galactosidse activity
1
0.5
polyA Reporter protein
Fig. 2. Effect of presence of both partners fused to Sup35C and Sup35NM on yeast phenotype. (A,B) Schematic representations of expected results when expressing Sup35NM– miniT (A) or Sup35NM–R3 (B). Endog en, endogenous. (C) Measure of W13 expression with a pair of interacting proteins or noninteracting proteins. (D) Test of the influence of P53 fused to Sup35C on the Sup35NM–miniT effect on W13 expression restoration.
PCA, in our system bait and prey proteins are in a translational fusion format. Our technology allows us to ensure the functionality of Sup35C fused to a given bait protein before pursuing the inter action studies. Y2H has been successfully used to identify protein– protein interactions in several model organisms. Nevertheless, the interaction in Y2H occurs in the nucleus, which is not the natural cellular compartment of many proteins of interest for interaction studies. In contrast, our system provides a direct detection in the cytoplasm, where the posttranslational modifications are impor tant for protein interaction. This technology should prove to be useful in the characterization of proteins as well as in partner identification, interaction map ping, and drug discovery applications. It may also be adapted in the development of a tool for use in drug discovery such as the screen ing of small molec ules, peptides, or intrabodies able to modulate intracytoplasmic interactions in a high-throughput screening format. For this purpose, other reporter genes and expression cas settes are currently optimized for use in studies that could be the basis of developing drugs for use in disorders often involving the pathol ogical deregulation of otherwise finely regulated cellular protein interactions. Acknowledgments We thank M. Blondel (U613-INSERM, Brest) for providing the yeast 74D694 strain and yeast expression plasmids and for engaging in helpful discussions at the beginning of this project. We thank MilleGen’s sequencing service for the quality of the results. We also thank our collaborators: M.J. Carles for techni cal support and A. Deflisque, C. Monnet, O. Dubreuil, F. Crozet, and P. Brune for discussions and critical reviews of the manu script.
References [1] J. Piehler, New methodologies for measuring protein interactions in vivo and in vitro, Curr. Opin. Struct. Biol. 15 (2005) 4–14. [2] T. Berggård, S. Linse, P. James, Methods for the detection and analysis of pro tein–protein interactions, Proteomics 7 (2007) 2833–2842. [3] S.B. Prusiner, Novel proteinac eous infectious particles cause scrapie, Science 216 (1982) 136–144. [4] M.D. Ter-Avanesyan, A.R. Dagkesamanskaya, V.V. Kushnirov, V.N. Smirnov, The SUP35 omnipotent suppressor gene is involved in the maintenance of the nonMendelian determinant [PSI+] in the yeast Saccharomyces cerevisiae, Genetics 137 (1994) 671–676. [5] I. Stansfield, K.M. Jones, V.V. Kushnirov, A.R. Dagkesamanskaya, A.I. Poznya kovski, S.V. Paushkin, C.R. Nierras, B.S. Cox, M.D. Ter-Avanesyan, M.F. Tuite, The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae, EMBO J. 14 (1995) 4365–4373. [6] A.L. Manogaran, K.T. Kirkland, S.W. Liebman, An engineered nonsense URA3 allele provides a versatile system to detect the presence, absence, and appear ance of the [PSI+] prion in Saccharomyces cerevisiae, Yeast 23 (2006) 141–147. [7] J.S. Butel, J.A. Lednicky, Cell and molecul ar biology of Simian virus 40: implica tions for human infections and disease, J. Natl. Cancer Inst. 91 (1999) 119–134. [8] E. Fanning, Simian virus 40 large T antigen: the puzzle, the pieces, and the emerging picture, J. Virol. 66 (1992) 1289–1293. [9] P.A. Ivanov, E.I. Lewitin, B.I. Shevelev, G.V. Fominov, J.A. Wojciechowska, A.H. Asadi Mobarhan, Y.V. Vertie, N.K. Yankovsky, A.B. Shevelev, Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of Botulinum neurotoxin in Saccharomyces cerevisiae, Res. Microbiol. 152 (2001) 27–35. [10] P. Zhou, I.L. Derkatch, S.W. Liebman, The relationship between visible intra cellular aggregates that appear after overexpression of Sup35 and the yeast prion-like elements [PSI(+)] and [PIN(+)], Mol. Microbiol. 39 (2001) 37–46. [11] S. Lindquist, S. Krobitsch, L. Li, N. Sondheimer, Investigating protein conforma tion-based inheritance and disease in yeast, Philos. Trans. R. Soc. Lond. B 356 (2001) 169–176. [12] E.E. Ganusova, L.N. Ozolins, S. Bhagat, G.P. Newnam, R.D. Wegrzyn, M.Y. Sher man, Y.O. Chernoff, Modulation of prion formation, aggregation, and toxicity by the actin cytoskeleton in yeast, Mol. Cell. Biol. 26 (2006) 617–629. [13] S. Fields, O. Song, A novel genetic system to detect protein–protein interac tions, Nature 340 (1989) 245–246. [14] N. Johnsson, A. Varshavsky, Split ubiquitin as a sensor of protein interactions in vivo, Proc. Natl. Acad. Sci. USA 91 (1994) 10340–10344.