Detecting Interactions of Membrane Proteins: The Split-Ubiquitin System.
Methods Mol Biol
; 1794: 49-60, 2018.
Article
em En
| MEDLINE
| ID: mdl-29855950
ABSTRACT
The in vivo analysis of protein-protein interactions (PPIs) is a critical factor for gaining insights into cellular mechanisms and their biological functions. To that end, a constantly growing number of genetic tools has been established, some of which are using baker's yeast (Saccharomyces cerevisiae) as a model organism. Here, we provide a detailed protocol for the yeast mating-based split-ubiquitin system (mbSUS) to study binary interactions among or with full-length membrane proteins in their native subcellular environment. The system is based on the reassembly of two autonomously non-functional ubiquitin moieties attached to proteins of interest (POIs) into a native-like molecule followed by the release of a transcription factor. Upon its nuclear import, the activation of reporter gene expression gives a visual output via growth on interaction-selective media. Additionally, we apply a modification of the classical split-ubiquitin technique called CytoSUS that detects interactions of non-membrane/soluble proteins in their full-length form via translational fusion of an ER membrane anchor.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Fatores de Transcrição
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Técnicas do Sistema de Duplo-Híbrido
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Proteínas de Saccharomyces cerevisiae
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Ubiquitina
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Mapeamento de Interação de Proteínas
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Proteínas de Membrana
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Alemanha