Abstract
Protein functions within cells frequently require they interact physically with a number of partner proteins to coordinate the appropriate biochemical processes. Mutational analysis has been quite useful for analyzing how the loss of a gene or protein impacts cell function or more specifically particular pathways. However, the genetics approach to studying gene function can be limited by not having the right mutations; for example because the mutant allele ablates all function, as is the case for deletion (null) alleles and most temperature-sensitive alleles. To dissect the relative contributions of a protein’s interactions, the researcher needs mutations that specifically affect one but not all of the protein’s interactions. In genetics parlance such mutations are called separation-of-function mutations. The yeast two-hybrid system has been exploited for two decades to identify protein-binding partners. Here we describe a fairly simple protocol, within reach of laboratories with molecular biology experience, for using the two-hybrid system to identify separation-of-function mutations.
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References
Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246
Chien CT, Bartel PL, Sternglanz R et al (1991) The two hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A 88:9578–9582
Amberg DC, Basart E, Botstein D (1995) Defining protein interactions with yeast actin in vivo. Nat Struct Biol 2:28–35
Amberg DC, Botstein D (1997) Obtaining structural information about protein complexes with the two-hybrid system. In: Bartel PL, Fields S (eds) The yeast two-hybrid system. Oxford University Press, New York
Leanna CA, Hannink M (1996) The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions. Nucleic Acids Res 24:3341–3347
Rodal AA, Tetreault JW, Lappalainen P et al (1999) Aip1p interacts with cofilin to disassemble actin filaments. J Cell Biol 145:1251–1264
Clark MG, Teply J, Haarer BK et al (2006) A genetic dissection of Aip1p’s interactions leads to a model for Aip1p-cofilin cooperative activities. Mol Biol Cell 17:1971–1984
Durfee T, Becherer K, Chen PL et al (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev 7:555–569
Amberg DC, Burke DJ, Strathern JN (2005) Methods in yeast genetics: a cold spring harbor laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Ma H, Kunes S, Schatz PJ et al (1987) Plasmid construction by homologous recombination in yeast. Gene 58:201–216
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Haarer, B., Amberg, D.C. (2014). Using Two-Hybrid Interactions to Identify Separation-of-Function Mutations. In: Smith, J., Burke, D. (eds) Yeast Genetics. Methods in Molecular Biology, vol 1205. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1363-3_9
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DOI: https://doi.org/10.1007/978-1-4939-1363-3_9
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