Poster abstracts
Poster number 101 submitted by Sarah Nock
Yeast engineered for enhanced identification of protein interaction networks using in vivo Proximity Labeling
Sarah L.H. Nock (Department of Genetics and Genome Sciences, Case Western Reserve University), Emily Parslow (Department of Genetics and Genome Sciences, Case Western Reserve University), Romeo Gallego (Bridges to the Baccalaureate Research Training Program, Cuyahoga Community College), Kristian E. Baker (Department of Genetics and Genome Sciences, Case Western Reserve University)
Abstract:
Proximity Labeling (PL) employing a biotin ligase fused to a protein of interest provides a powerful approach to identify protein interaction networks in the cell and offers a number of advantages over traditional approaches used to map protein-protein interactions. Protein ‘partners’ biotinylated in vivo are easily captured using streptavidin affinity purification and identified via mass spectrometry; however, naturally occurring, endogenous biotinylated proteins present in all cells represent a common contaminant in every PL experiment. Due to their general abundance, these proteins can obscure detection of lowly abundant protein partners present in samples, thereby reducing the overall sensitivity of this assay.
To mitigate this important limitation in PL experiments in yeast, we have used CRISPR-Cas9 genome editing to introduce an epitope-tag into the genes expressing these endogenous biotinylated proteins as a means to facilitate immunodepletion of these proteins from samples prior to streptavidin affinity purification. Here, we describe in detail the construction and characterization of this yeast strain and demonstrate how this approach significantly enhances the signal-to-noise level in PL experiments performed to investigate protein interactions involved in nonsense-mediated mRNA decay (NMD). Our data indicate that this strain is a valuable resource for researchers employing PL in yeast in their labs
References:
Branon, T., Bosch, J., Sanchez, A. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36, 880–887 (2018). https://doi.org/10.1038/nbt.4201
Mellacheruvu, D., Wright, Z., Couzens, A. et al. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nat Methods 10, 730–736 (2013). https://doi.org/10.1038/nmeth.2557
Novarina, D., Koutsoumpa, A., Milias-Argeitis, A. A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast. STAR Protocols 3, 101358 (2022). https://doi.org/10.1016/j.xpro.2022.101358
Keywords: proximity labeling, protein-protein interactions, yeast, NMD, gene editing