Identification of Genes Important for the Physical Interaction between Protein Pairs through Reverse PCA (rPCA)

Ifat Lev1, Marina Volpe1, Shay Ben‐Aroya1

1 Faculty of Life Sciences Bar‐Ilan University, Ramat‐Gan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.15
DOI:  10.1002/0471143030.cb1715s64
Online Posting Date:  September, 2014
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Abstract

Cells contain many important protein complexes involved in performing and regulating structural, metabolic, and signaling functions. Understanding physical and functional interactions between proteins in living systems is of vital importance in biology. The importance of protein‐protein interactions (PPIs) has led to the development of several powerful methodologies and techniques to detect them. All of this information has enabled the creation of large protein‐interaction networks. One important challenge in biology is to understand how protein complexes respond to genetic perturbations. Here we describe a systematic genetic assay termed “reverse PCA,” which allows the identification of genes whose products are required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the PPI of interest can be detected by resistance to the drug methotrexate, in the context of the protein‐fragment complementation assay (PCA). By combining the synthetic genetic array (SGA) technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans‐acting mutations that disrupt the physical interaction of interest. The identification of such mutants is valuable for unraveling important regulatory mechanisms, and for defining the response of the protein interactome to specific perturbations. Curr. Protoc. Cell Biol. 64:17.15.1‐17.15.11. © 2014 by John Wiley & Sons, Inc.

Keywords: protein‐protein interaction (PPI); protein complementation assay (PCA); Saccharomyces cerevisiae; synthetic genetic array (SGA); high‐throughput technologies

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Haploid strains harboring the specific fusion proteins of interest (X::F[1,2]‐NatMX and Y::F[3]‐HygB), can be selected from two commercially available collections (Open Biosystems, cat. no. YSC5849); also see recipe for yeast strains
  • Media:
    • YEPD medium (see recipe for Media)
    • YEPD+clonNAT+G418 (see recipe for Media)
    • SPO medium (see recipe for Media)
    • SD/MSG medium (see recipe for Media)
    • SD/MSG+clonNAT+HygB+G418 medium (see recipe for Media)
    • SD/MSG+clonNAT+HygB+G418+MTX medium (see recipe for Media)
  • 30°C bacteriological incubator
  • Disposable and sterilized media plates and plastic pads of pins with standard footprint dimensions specifically designed for high‐throughput pinning using the Rotor HDA (Singer Instruments, http://www.singerinst.co.uk)
  • Robotic pin tool: the yeast colony arrayer robot (Singer Rotor HDA bench‐top robot; Singer Instruments, http://www.singerinst.co.uk) is programmed to manipulate yeast cell arrays and can be used for all the systematic pinning steps described below. The rotor uses disposable plastic replicator pads that can support agar pinning at densities of 96, 384, 768, and 1536 formats.
  • Software for data analysis: the “Balony” software allows image analysis and data inspection for agar plates generated in high‐throughput yeast genetics and genomics experiments. The software was developed by Christopher Loewen's lab (The University of British Columbia; Young and Loewen, ). The following link allows free access to download the software package: https://code.google.com/p/balony/downloads/list.
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Figures

Videos

Literature Cited

Literature Cited
  Babu, M., Krogan, N.J., Awrey, D.E., Emili, A., and Greenblatt, J.F. 2009. Systematic characterization of the protein interaction network and protein complexes in Saccharomyces cerevisiae using tandem affinity purification and mass spectrometry. Methods Mol. Biol. 548:187‐207.
  Costanzo, M., Baryshnikova, A., Bellay, J., Kim, Y., Spear, E.D., Sevier, C.S., Ding, H., Koh, J.L., Toufighi, K., Mostafavi, S., Prinz, J., St Onge, R.P., VanderSluis, B., Makhnevych, T., Vizeacoumar, F.J., Alizadeh, S., Bahr, S., Brost, R.L., Chen, Y., Cokol, M., Deshpande, R., Li, Z., Lin, Z.Y., Liang, W., Marback, M., Paw, J., San Luis, B.J., Shuteriqi, E., Tong, A.H., van Dyk, N., Wallace, I.M., Whitney, J.A., Weirauch, M.T., Zhong, G., Zhu, H., Houry, W.A., Brudno, M., Ragibizadeh, S., Papp, B., Pal, C., Roth, F.P., Giaever, G., Nislow, C., Troyanskaya, O.G., Bussey, H., Bader, G.D., Gingras, A.C., Morris, Q.D., Kim, P.M., Kaiser, C.A., Myers, C.L., Andrews, B.J., and Boone, C. 2010. The genetic landscape of a cell. Science 327:425‐431.
  Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau‐Danila, A., Anderson, K., Andre, B., Arkin, A.P., Astromoff, A., El‐Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Curtiss, M., Davis, K., Deutschbauer, A., Entian, K.D., Flaherty, P., Foury, F., Garfinkel, D.J., Gerstein, M., Gotte, D., Guldener, U., Hegemann, J.H., Hempel, S., Herman, Z., Jaramillo, D.F., Kelly, D.E., Kelly, S.L., Kotter, P., LaBonte, D., Lamb, D.C., Lan, N., Liang, H., Liao, H., Liu, L., Luo, C., Lussier, M., Mao, R., Menard, P., Ooi, S.L., Revuelta, J.L., Roberts, C.J., Rose, M., Ross‐Macdonald, P., Scherens, B., Schimmack, G., Shafer, B., Shoemaker, D.D., Sookhai‐Mahadeo, S., Storms, R.K., Strathern, J.N., Valle, G., Voet, M., Volckaert, G., Wang, C.Y., Ward, T.R., Wilhelmy, J., Winzeler, E.A., Yang, Y., Yen, G., Youngman, E., Yu, K., Bussey, H., Boeke, J.D., Snyder, M., Philippsen, P., Davis, R.W., and Johnston, M. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387‐391.
  Ho, Y., Gruhler, A., Heilbut, A., Bader, G.D., Moore, L., Adams, S.L., Millar, A., Taylor, P., Bennett, K., Boutilier, K., Yang, L., Wolting, C., Donaldson, I., Schandorff, S., Shewnarane, J., Vo, M., Taggart, J., Goudreault, M., Muskat, B., Alfarano, C., Dewar, D., Lin, Z., Michalickova, K., Willems, A.R., Sassi, H., Nielsen, P.A., Rasmussen, K.J., Andersen, J.R., Johansen, L.E., Hansen, L.H., Jespersen, H., Podtelejnikov, A., Nielsen, E., Crawford, J., Poulsen, V., Sorensen, B.D., Matthiesen, J., Hendrickson, R.C., Gleeson, F., Pawson, T., Moran, M.F., Durocher, D., Mann, M., Hogue, C.W., Figeys, D., and Tyers, M. 2002. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180‐183.
  Jares‐Erijman, E.A. and Jovin, T.M. 2006. Imaging molecular interactions in living cells by FRET microscopy. Curr. Opin. Chem. Biol. 10:409‐416.
  Lev, I., Volpe, M., Goor, L., Levinton, N., Emuna, L., and Ben‐Aroya, S. 2013. Reverse PCA, a systematic approach for identifying genes important for the physical interaction between protein pairs. PLoS Genetics 9:e1003838.
  Michnick, S.W., Ear, P.H., Landry, C., Malleshaiah, M.K., and Messier, V. 2010. A toolkit of protein‐fragment complementation assays for studying and dissecting large‐scale and dynamic protein‐protein interactions in living cells. Methods Enzymol. 470:335‐368.
  Remy, I., Galarneau, A., and Michnick, S.W. 2002. Detection and visualization of protein interactions with protein fragment complementation assays. Methods Mol. Biol. 185:447‐459.
  Remy, I., Campbell‐Valois, F.X., and Michnick, S.W. 2007. Detection of protein‐protein interactions using a simple survival protein‐fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc. 2:2120‐2125.
  Tarassov, K., Messier, V., Landry, C.R., Radinovic, S., Serna Molina, M.M., Shames, I., Malitskaya, Y., Vogel, J., Bussey, H., and Michnick, S.W. 2008. An in vivo map of the yeast protein interactome. Science 320:1465‐1470.
  Tong, A.H. and Boone, C. 2006. Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol. Biol. 313:171‐192.
  Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., 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., and Rothberg, J.M. 2000. A comprehensive analysis of protein‐protein interactions in Saccharomyces cerevisiae. Nature 403:623‐627.
  Young, B.P. and Loewen, C.J. 2013. Balony: A software package for analysis of data generated by synthetic genetic array experiments. BMC Bioinform. 14:354.
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