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, used to date, is the two-hybrid system. Although proven very powerful, the two-hybrid system exhibits several inherent problems and limitations. Recently, two alternative systems have been described that take advantage of the fact that localization of signal transduction effectors to the inner leaflet of the plasma membrane is absolutely necessary for yeast viability. These effectors can either be the Ras guanyl nucleotide exchange factor or Ras itself. The yeast strain used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl nucleotide exchange factor, CDC25. Membrane localization of these effectors is achieved via protein +/- protein interaction. 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 recruitment systems.
Aronheim
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Department of Molecular Genetics, Rappaport Family Institute for Research in the Medical Sciences, Bat-Galim, Haifa 31096, Israel. aronheim@tx.technion.ac.il
D018797:Cell Cycle Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D015398:Signal Transduction; D013696:Temperature; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020666:ras-GRF1 / Q000378:metabolism
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020666:ras-GRF1