Protein recruitment systems for the analysis of protein +/- protein interactions

The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the analysis of protein +/- protein interactions. The most common system, u sed to date, is the two-hybrid system. Although proven very powerful, the t wo-hybrid system exhibits several inherent problems and limitations. Recent ly, two alternative systems have been described that take advantage of the fact that localization of signal transduction effecters to the inner leafle t of the plasma membrane Is absolutely necessary for yeast viability. These effecters can either be the Ras guanyl nucleotide exchange factor or Ras i tself. The yeast strain used in both systems is a temperature-sensitive mut ant in the yeast Ras guanyl nucleotide exchange factor, CDC25. Membrane loc alization of these effecters is achieved via protein +/- protein interactio n. Each system can be used to test interaction between known protein pairs, as well as for isolation of novel protein interactions. Described here are the scientific and technical steps to be considered for both protein recru itment systems. (C) 2001 Academic Press.