Cell growth selection system to detect extracellular and transmembrane protein interactions.
The interplay among extracellular and cell surface proteins, such as the interactions between ligands and receptors or between antigens and antibodies, is involved in a multitude of physiological and pathological phenomena. In the oxidizing milieu of the secretory pathway in eukaryotic cells, many extracellular proteins build disulfide bonds that significantly contribute to their correct folding and structural stability. Thus, conventional yeast two-hybrid interaction assays, which occur in the reducing intracellular environment, might not be adequate to detect extracellular protein-protein interactions. We have exploited the properties of yeast Ire1p, a type I endoplasmic reticulum (ER) membrane protein involved in the unfolded protein response (UPR) as a dimerization-activated receptor, to develop a novel system for the detection and study of interactions between extracellular and/or membrane proteins. In our system, named SCINEX-P (screening for interactions between extracellular proteins), proteins of interest were fused to truncated Ire1p so as to substitute its N-terminal lumenal domain (NLD). Specific interaction between two partners caused dimerization of the Ire1p moiety, which, through the endogenous UPR signalling pathway, led to activation of transcription of genes that permit cell growth under selective conditions.
Urech
David M
DM
ESBATech AG, Wagistrasse 21, CH-8952 Zürich-Schlieren, Switzerland. urech@esbatech.com
Lichtlen
Peter
P
Barberis
Alcide
A
D050976:Basic-Leucine Zipper Transcription Factors; D002455:Cell Division; D003114:Colony-Forming Units Assay / Q000379:methods*; D019281:Dimerization; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics
D050976:Basic-Leucine Zipper Transcription Factors; C091341:HAC1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C082863:IRE1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases