Identification of protein-protein interactions between integral
membrane proteins.
John P. Miller (1), Safia Thaminy (2), Igor Stagljar (3), Stanley
Fields (4)
(1) Genome Sciences, University of Washington, 1959 NE Pacific St.,
Seattle, WA 98195-7730, United States of America;
(2) Institute of Veterinary Biochemistry and Molecular Biology,
University of Zurich-Irchel, Winterthurerstrasse 190, CH-8057 Zurich,
Switzerland;
(3) Institute of Veterinary Biochemistry and Molecular Biology,
University of Zurich-Irchel and Dualsystems Biotech, Inc.,
Winterthurerstrasse 190, CH-8057 Zurich, Switzerland;
(4) Howard Hughes Medical Institute, Department of Genome Sciences and
Department of Medicine, University of Washington, Seattle, WA 98195-7730
We have generated an assay system to analyze interactions between
integral membrane proteins in the hope that such information can provide
clues as to the functions of these proteins. To this end, we created an
array of yeast colonies each expressing a different integral membrane
protein-fusion competent for use in the split-ubiquitin system
originally developed by Johnsson and Varshavsky (PNAS
91:10340-10344 (1994)). The system is based on the ability of
interacting proteins to bring two fragments of ubiquitin into close
proximity such that they are acted on by ubiquitin-specific proteases.
Proteolysis releases a reporter protein, in our case a transcription
factor that then enters the nucleus to activate reporter genes (Stagljar
et al. PNAS 95:5187-5192 (1998)). We selected proteins
annotated in the Yeast Proteome Database as 'integral membrane' to place
into the array. For each of the ~700 proteins in the array we generated
constructs designed to make hybrids in two forms: with the membrane
protein amino-terminal or carboxyl-terminal to one of the halves of
ubiquitin. In a pilot project, we screened an array of ~40 membrane
proteins against components of the OST complex, ER-associated
degradation pathway, and translocation machinery, and we are planning to
further study these and other membrane complexes against the larger
array.
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