Haarer Brian, Amberg David C
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse, NY, 13210, USA.
Methods Mol Biol. 2014;1205:131-42. doi: 10.1007/978-1-4939-1363-3_9.
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.
