Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex

dc.contributor.authorPfannenstein, Jeffrey
dc.contributor.authorTyryshkin, Misha
dc.contributor.authorGulden, Moira E.
dc.contributor.authorDoud, Emma H.
dc.contributor.authorMosley, Amber L.
dc.contributor.authorReese, Joseph C.
dc.contributor.departmentBiochemistry and Molecular Biology, School of Medicine
dc.date.accessioned2024-12-06T11:13:47Z
dc.date.available2024-12-06T11:13:47Z
dc.date.issued2024
dc.description.abstractThe modified Escherichia coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37°C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a thorough comparison of these variations of biotin ligases and the development of versatile tools for conducting these experiments in Saccharomyces cerevisiae would benefit the community. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive labeling of proteins even in the absence of exogenous biotin, due to biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.
dc.eprint.versionFinal published version
dc.identifier.citationPfannenstein J, Tyryshkin M, Gulden ME, Doud EH, Mosley AL, Reese JC. Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex. G3 (Bethesda). 2024;14(11):jkae221. doi:10.1093/g3journal/jkae221
dc.identifier.urihttps://hdl.handle.net/1805/44792
dc.language.isoen_US
dc.publisherOxford University Press
dc.relation.isversionof10.1093/g3journal/jkae221
dc.relation.journalG3
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttps://creativecommons.org/licenses/by/4.0
dc.sourcePMC
dc.subjectBioID
dc.subjectProximity labeling
dc.subjectCcr4-Not
dc.subjectmRNA decay
dc.titleCharacterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex
dc.typeArticle
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