Bimolecular Fluorescence Complementation to Assay the Interactions of Ubiquitylation Enzymes in Living Yeast Cells.
Ubiquitylation is a versatile posttranslational protein modification catalyzed through the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). These enzymes form transient complexes with each other and their modification substrates and determine the nature of the ubiquitin signals attached to their substrates. One challenge in the field of protein ubiquitylation is thus to identify the E2-E3 pairs that function in the cell. In this chapter, we describe the use of bimolecular fluorescence complementation to assay E2-E3 interactions in living cells, using budding yeast as a model organism.
Department of Genetics and Cell Physiology, Institute of Experimental Biology, University of Wroclaw, Kanonia 6/8, Wroclaw, 50-328, Poland.
Ewa
E
Blaszczak
Centre National de la Recherche Scientifique, UMR 6290, Rennes, France.|Institut de Génétique et Développement de Rennes, Université de Rennes 1, Rennes, France.
Claude
C
Prigent
Centre National de la Recherche Scientifique, UMR 6290, Rennes, France. gwenael.rabut@inserm.fr.|Institut de Génétique et Développement de Rennes, Université de Rennes 1, Rennes, France. gwenael.rabut@inserm.fr.|Institute of Genetics and Development of Rennes, UMR 6290 CNRS-UR1, 2 avenue du Professeur Léon Bernard, CS 34317, Rennes, Cedex, 35043, France. gwenael.rabut@inserm.fr.
Gwenaël
G
Rabut
D001681:Biological Assay / Q000379:methods*; D005453:Fluorescence; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology*
D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
BiFC; Linear unmixing; Living cell; Microscopy; Protein-fragment complementation assay; Protein–protein interactions; Saccharomyces cerevisiae; Ubiquitin; Ubiquitin ligase; Ubiquitin-conjugating enzyme