A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo.
A detection system for interactions between membrane proteins in vivo is described. The system is based on split-ubiquitin [Johnsson, N. & Varshavsky, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10340-10344]. Interaction between two membrane proteins is detected by proteolytic cleavage of a protein fusion. The cleavage releases a transcription factor, which activates reporter genes in the nucleus. As a result, interaction between membrane proteins can be analyzed by the means of a colorimetric assay. We use membrane proteins of the endoplasmic reticulum as a model system. Wbp1p and Ost1p are both subunits of the oligosaccharyl transferase membrane protein complex. The Alg5 protein also localizes to the membrane of the endoplasmic reticulum, but does not interact with the oligosaccharyltransferase. Specific interactions are detected between Wbp1p and Ost1p, but not between Wbp1p and Alg5p. The new system might be useful as a genetic and biochemical tool for the analysis of interactions between membrane proteins in vivo.
Stagljar
I
I
Institute of Veterinary Biochemistry, University Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Korostensky
C
C
Johnsson
N
N
te Heesen
S
S
D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D005822:Genetic Vectors; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014166:Transferases / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases; D014452:Ubiquitins / Q000737:chemistry*
D017405:Herpes Simplex Virus Protein Vmw65; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases