RESUMO
Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.
Assuntos
Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Quitina Sintase/metabolismo , Detergentes , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Espectrometria de Massas , Proteínas de Membrana/análise , Proteínas de Membrana/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.