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

ABSTRACT: The modified E. 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 comparison of these variations of biotin ligases has not been reported in Saccharomyces cerevisiae. 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 activity from 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.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)

SUBMITTER: Joseph C. Reese   Amber L. Mosley  

PROVIDER: MSV000094663 | MassIVE | Thu May 02 12:59:00 BST 2024

REPOSITORIES: MassIVE