A novel approach for the identification of protein-protein interaction with integral membrane proteins.
Protein-protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein-protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein-protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein-protein interactions with high specificity and selectivity, where other methods fail.
Hubsman
M
M
Department of Molecular Genetics and the Rappaport Family Institute for Research in the Medical Sciences and the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, PO Box 9649, Bat-Galim, Haifa 31096, Israel.
Yudkovsky
G
G
Aronheim
A
A
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010902:Pituitary Gland / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013379:Substrate Specificity; D014170:Transformation, Genetic; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020666:ras-GRF1 / Q000235:genetics
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D008715:Methionine; D018631:ras Proteins; D005690:Galactose