Dipartimento di Biologia, Università Tor Vergata, via della Ricerca Scientifica, 00133 La Romanina (Roma), Italy1
Centro Acidi Nucleici del CNR, Roma, Italy2
Author for correspondence: L. Paolozzi. Tel: +39 6 72594674. Fax: +39 6 2023500. e-mail: Paolozzi{at}bio.uniroma2.it
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ABSTRACT |
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Keywords: proteinprotein interaction, cell division proteins, phage repressor, ß-galactosidase assay
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INTRODUCTION |
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To extend this method to the study of protein heterodimerization, we constructed a chimeric operator formed by the two hemi-sites of the phage P22 and phage 434 operators. This operator can be recognized and bound only by a hybrid repressor formed by two chimeric monomers; one with the N-terminal portion of phage 434 and the other with that of phage P22. The C-terminal domains of both of them are composed of heterologous proteins (or protein domains) whose interaction ability is under investigation. Only those proteins that mediate efficient dimerization of the two chimeric repressor monomers in vivo permit the formation of a functional repressor able to bind the P22434 hybrid operator and shut off the synthesis of a downstream reporter gene.
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METHODS |
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Construction of a cI N-terminal fragment with phage P22 operator specificity.
Plasmid pC172 is a pC132 (Longo et al., 1995 ) derivative in which the
cI N-terminal fragment was substituted with a 281 bp fragment containing a phage 434 cI N-terminal fragment. In order to obtain the binding specificity of phage P22 (Wharton & Ptashne, 1987
), we performed a PCR site-specific mutagenesis using the synthetic oligonucleotide 5'-GTTTTACCTCTTTCGAGCTGCGATATAGAAACATTGGAAGTCCCCA-3' (underlining indicates the codons that were changed in order to switch the specificity: T28S, Q29N, Q30V, E33S, N37R). The resulting plasmids were verified by sequencing.
Construction of pcIP22 and pcI434 derivatives.
The various derivatives of pcIP22 and pcI434 reported in Table 1 were constructed by cloning the DNA of the gene of interest obtained by PCR amplification using the following pairs of 28 bp synthetic oligonucleotides into the pcI plasmid digested with SalI and BamHI: forward, 5'-GCGTCGACC plus 19 specific nucleotides of the gene sequence starting from ATG; reverse, 5'-CGGGATCC plus 19 specific nucleotides of the gene sequence starting from the stop codon.
In the case of Max and Myc, only the bHLH-LZ domains (corresponding to amino acids 351439 and 22105, respectively) were cloned into the pcI plasmids. The synthetic oligonucleotides used were as follows: for Max, 5'-GAGGTGGAGTCGACCGCTGACAAACGG-3', 5'-CAGTTGGGGGATCCACTACGCCTTCTCCAG-3'; for Myc, 5'-GTCCTCGTCGACCGAGGAGAATGTC-3', 5'-TCCTTAGGATCCCTTACGCACAAGAG-3'.
Dimerization assay: ß-galactosidase assay.
Assay of ß-galactosidase activity was performed on bacterial cultures grown in LuriaBertani medium supplemented with 1x10-4 M IPTG to OD600 0.5, as described by Miller (1972) .
General microbiological and recombinant DNA techniques.
Standard microbiological and recombinant DNA techniques were as described by Miller (1972) and Sambrook et al. (1989)
, respectively.
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RESULTS |
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Validation of the assay
The C-terminal domain of the phage 434 repressor was cloned in-frame downstream of the N-terminal portion of the repressor in both pcI434 and pcIP22, giving plasmids pcI434-434 and pcIP22-434, respectively. The pcI plasmids, with or without the 434 C-terminal domain, were inserted by transformation into strain R721, and the ß-galactosidase activity was determined after induction with 1x10-4 M IPTG (Table 2a).
As shown in Table 2, neither the presence of one of the pcI plasmids (pcI434 or pcIP22), nor the presence of both modifies the quantity (Miller units) of ß-galactosidase produced by R721: in every case, the residual activity was of the order of 100%. Analogously, when pcI434-434 or pcIP22-434 was inserted in R721, the amount of ß-galactosidase produced was not substantially changed.
When plasmid pcI434-434 and pcIP22-434 were both present in R721, the result was dramatically different. In this case, the functional hybrid repressor formed was able to recognize the chimeric operator and shut off lacZ gene transcription and so the ß-galactosidase activity dropped to 94 Miller units (3.7% of residual activity). This result was confirmed without the addition of IPTG, 1107 Miller units of ß-galactosidase (44.1% of residual activity) being produced.
Homo- and heterodimerization assay
To test the efficiency of our two-hybrid assay in detecting homo- and heterodimerization, we assayed various pairs of proteins or protein domains both prokaryotic and eukaryotic.
The ability of this two-hybrid system to reveal homodimerization, already demonstrated with the C-terminal portion of phage 434 repressor, was confirmed also when the N-terminal domains of both phages were fused with the cat gene (Table 2b).
The other parts of Table 2 deal with heterodimerization. As shown, the assay works both when large proteins are cloned in-frame with the cI N-terminal fragment, as demonstrated by the interaction between GyrA (97 kDa) and GyrB (90 kDa) (Table 2c
) and when small interaction domains, such as the bHLH-LZ domains of Myc and Max, both of which are about 10 kDa in size (Table 2e
). In addition, these two examples show that there is no difference between prokaryotic and eukaryotic proteins.
Particularly interesting is the characterization of FtsZ and FtsA interaction (Table 2d). We observed that it is not important which protein is cloned in the high-copy-number plasmid; in fact, the amounts of ß-galactosidase are similar when ftsZ was cloned downstream of the N terminus of either 434 or P22. Moreover, the behaviour of plasmids pcIP22-ftsZ
Nter and pcIP22-ftsZ
Cter shows that the reduction in ß-galactosidase activity is observable only when the first plasmid is present in strain R721 together with pcI434-ftsA, confirming that FtsA interacts with the C-terminal domain of FtsZ (Wang et al., 1997
).
FtsZMinC interaction
Recent results (Hu & Lutkenhaus, 2000 ) showed that MinC, a component of the min system that prevents FtsZ from assembling at polar sites during bacterial cell division, consists of two independently functioning domains. The C-terminal domain is responsible for MinC localization through interaction with MinD, whilst the N-terminal domain inhibits FtsZ assembly, possibly by a direct interaction.
We studied this suggested interaction between MinC and FtsZ with the 434P22 two-hybrid system. The results (Table 2f) indicate that MinC and FtsZ interact in vivo, though the residual ß-galactosidase activity, higher than in all the other cases studied, might suggest that this interaction could be either weak or only transient.
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DISCUSSION |
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These considerations prompted the development of a number of alternative approaches that could allow these limitations to be overcome, or at least complement the yeast two-hybrid method. Most of these are carried out in E. coli and are based on a variation of the two-hybrid approach (Hu et al., 1990 ; Dove et al., 1997
; Karimova et al., 1998
, 2000
; Kornacker et al., 1998
). Aside from the considerations above, the ability to transpose the power of the yeast two-hybrid method into E. coli would be an advantage per se because of the ease of manipulation of this organism and because of the efficiency of transformation (which can be up to 2 logs higher than that in yeast).
Recently, it was shown that a strategy based on the reconstruction of mouse dihydropholate reductase can be efficiently used to perform a library-against-library selection in order to detect pairs of peptides that can form a leucine zipper (Pelletier et al., 1999 ). These methods based on the reconstitution of an enzymic activity, however, suffer from the drawback that the two complementing moieties need to be placed close to each other in a topologically correct position.
Hu et al. (1990) have shown that the
repressor can be effectively used as a reporter to detect dimeric proteins. This approach was used with several proteins and it is therefore likely that (like the yeast two-hybrid method) it is relatively topology-insensitive. In this work, we have extended this method by making it amenable to the detection of proteins that form a complex. We have tested this new approach with several interacting proteins ranging in size from less than 100 to more than 800 amino acids, and, to date, we have not been able to detect any size or topology limit. Furthermore, the system proved itself very sensitive, since we were able to identify the interaction between the prokaryotic proteins FtsZ and MinC an interaction that scored as negative in the yeast two-hybrid system (Hu & Lutkenhaus, 2000
). These two proteins bind with a very low affinity that is possibly below the lower limit of the yeast two-hybrid system. However, this result does not prove that our method is, in general, more sensitive than the yeast two-hybrid system. In fact, it is possible that in this case the low absolute affinity is compensated for by the recruitment and concentration of MinC to the membrane by association with MinD.
At the moment, the lambdoid repressor dimerization strategy that we have developed is just a convenient and promising alternative to the various two-hybrid methods that are being developed to study proteinprotein interactions. Here, we have tested ligands with a wide range of size and binding affinities, and we are in the process of developing a selection protocol in order to use this experimental setting for proteome analysis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Di Lallo, G., Anderluzzi, D., Ghelardini, P. & Paolozzi, L. (1999a). FtsZ dimerization in vivo. Mol Microbiol 2, 265-274.
Di Lallo, G., Ghelardini, P. & Paolozzi, L. (1999b). Two-hybrid assay: construction of an Escherichia coli system to quantify homodimerization ability in vivo. Microbiology 145, 1485-1490.[Abstract]
Dove, S. L., Joung, J. K. & Hochschild, A. (1997). Activation of prokaryotic transcription through arbitrary proteinprotein contacts. Nature 386, 627-630.[Medline]
Fields, S. & Song, O. (1989). A novel genetic system to detect proteinprotein interactions. Nature 340, 245-246.[Medline]
Hanes, J. & Pluckthun, A. (1999). In vitro selection methods for screening of peptide and protein libraries. Curr Top Microbiol Immunol 243, 107-122.[Medline]
Hu, Z. & Lutkenhaus, J. (2000). Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ. J Bacteriol 182, 3965-3971.
Hu, J. C., OShea, E. K., Kim, P. S. & Sauer, R. T. (1990). Sequence requirements for coiled-coils: analysis with repressor-GCN4 leucine zipper fusions. Science 250, 1400-1403.[Medline]
Karimova, G., Pidoux, J., Ullmann, A. & Ladant, D. (1998). A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 95, 5752-5756.
Karimova, G., Ullmann, A. & Ladant, D. (2000). A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli. Methods Enzymol 328, 59-73.[Medline]
Kornacker, M. G., Remsburg, B. & Menzel, R. (1998). Gene activation by the AraC protein can be inhibited by DNA looping between AraC and a LexA repressor that interacts with AraC: possible applications as a two-hybrid system. Mol Microbiol 30, 615-624.[Medline]
Longo, F., Marchetti, M. A., Castagnoli, L., Battaglia, P. A. & Gigliani, F. (1995). A novel approach to protein-protein interaction: complex formation between the p53 tumour suppressor and the HIV Tat proteins. Biochem Biophys Res Commun 206, 326-334.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Pelletier, J. N., Arndt, K. M., Pluckthun, A. & Michnick, S. W. (1999). An in vivo library-versus-library selection of optimized protein-protein interactions. Nat Biotechnol 17, 683-690.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Vidal, M. & Legrain, P. (1999). Yeast forward and reverse n-hybrid systems. Nucleic Acids Res 27, 919-929.
Wang, X., Huang, J., Mukherjee, A., Cao, C. & Lutkenhaus, J. (1997). Analysis of the interaction of FtsZ with itself, GTP, and FtsA. J Bacteriol 179, 5551-5559.[Abstract]
Webster, C., Merryweather, C. & Brammar, W. (1992). Efficient repression by a heterodimeric repressor in Escherichia coli. Mol Microbiol 6, 371-377.[Medline]
Wharton, R. P. & Ptashne, M. (1987). A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact. Nature 326, 888-891.[Medline]
Received 6 December 2000;
revised 8 February 2001;
accepted 14 February 2001.