Studying Protein Ubiquitylation in Yeast.
Ubiquitylation is a reversible posttranslational modification that is critical for most, if not all, cellular processes and essential for viability. Ubiquitin conjugates to substrate proteins either as a single moiety (monoubiquitylation) or as polymers composed of ubiquitin molecules linked to each other with various topologies and structures (polyubiquitylation). This contributes to an elaborate ubiquitin code that is decrypted by specific ubiquitin-binding proteins. Indeed, these different types of ubiquitylation have different functional outcomes, notably affecting the stability of the substrate, its interactions, its activity, or its subcellular localization. In this chapter, we describe protocols to determine whether a protein is ubiquitylated, to identify the site that is ubiquitylated, and provide direction to study the topology of the ubiquitin modification, in the yeast Saccharomyces cerevisiae.
Institut Jacques Monod, UMR 7592 CNRS/Univ. Paris Diderot, Sorbonne Paris Cité, Paris, France.
Junie
J
Hovsepian
Institut Jacques Monod, UMR 7592 CNRS/Univ. Paris Diderot, Sorbonne Paris Cité, Paris, France.|Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, 02115, USA.
Michel
M
Becuwe
Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
Oded
O
Kleifeld
Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Michael H
MH
Glickman
Institut Jacques Monod, UMR 7592 CNRS/Univ. Paris Diderot, Sorbonne Paris Cité, Paris, France. sebastien.leon@ijm.fr.|Membrane Trafficking, Ubiquitin and Signaling Lab, Institut Jacques Monod, UMR7592 CNRS/Université Paris-Diderot, 15 Rue Hélène Brion, Bât. Buffon, 75205, Paris Cedex 13, France. sebastien.leon@ijm.fr.
Sébastien
S
Léon
D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology*
D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D057149:Ubiquitinated Proteins
Histidine-tagged ubiquitin purification; Immunoprecipitation in denaturing conditions; Ubiquitin; Ubiquitin chain topology; Ubiquitylation site mapping; Yeast