Proximity-dependent biotinylation mediated by TurboID to identify protein–protein interaction networks in yeast

Abstract

The use of proximity-dependent biotinylation assays coupled to mass spectrometry (PDB-MS) has changed the field of protein-protein interaction studies. However, despite the recurrent and successful use of BioID-based protein-protein interactions screening in mammalian cells, the implementation of PDB-MS in yeast has not been effective. Here, we report a simple and rapid approach in yeast to effectively screen for proximal and interacting proteins in their natural cellular environment by using TurboID, a recently described version of the BirA biotin ligase. Using the protein arginine methyltransferase Rmt3 and the RNA exosome subunits, Rrp6 and Dis3, the application of PDB-MS in yeast by using TurboID was able to recover protein-protein interactions previously identified using other biochemical approaches and provided new complementary information for a given protein bait. The development of a rapid and effective PDB assay that can systematically analyze protein-protein interactions in living yeast cells opens the way for large-scale proteomics studies in this powerful model organism.

TurboID-mediated proximity labelling of cytoophidium proteome inDrosophila

“…Furthermore, we found that the biotinylated proteins had similar distribution patterns to the wild type, or disrupted cytoophidia, which suggested that the proximate proteins of CTPS-TbID/CTPS H355A -TbID were easily biotinylated ( Fig 3C). In the absence of exogenous biotin, some weak labelling signals were also detected by streptavidin-cy3 ( Fig 3C), which was expected because TbID has great efficiency and consumes endogenous biotin in cells in order to function, as was reported in previous studies (Branon et al, 2018;Larochelle et al, 2019).…”

“…Then, we analyzed the biotinylated proteins adjacent to CTPS-TbID or CTPS H355A -TbID by hierarchical clustering, and our results revealed the differences among the proteome between normal CTPS cytoophidium and disrupted cytoophidium groups (Fig 4B). To assess the relative abundance of the characterized proteins, we analyzed MS/MS counts plotted against the protein sequence coverage (percentage of amino acid of a protein characterized by MS) by calculating the counts of all peptides matching to a specific protein (Larochelle et al, 2019;Schwanhausser et al, 2011). In addition to CTP synthase, another two known CTPS-interacting proteins (awd, ras) were found in our assay, and, as expected, CTPsyn was determined as a top hit ( Fig 4C).…”

“…Because of the high labelling efficiency and lower temperature requirement, TurboID and miniTurbo have been utilised to profile interaction networks in S. pombe, N. benthamiana, and Arabidopsis (Larochelle et al, 2019;Mair et al, 2019;.…”

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

“…However, recent studies showed that this time might be adapted depending on the organism studied. While 10 min is sufficient in mammalian cells, TurboID‐mediated biotinylation requires about 30 min to 3 h in yeast, 4 h in flies, 4 h to several days in worms, and 12 h in plants . Optimization of the TurboID labeling time should therefore be done for each organism, especially to prevent toxicity via chronic endogenous biotinylation or endogenous biotin consumption due to the high activity of these enzymes …”

Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector–Host Protein–Protein Interactions

“…These elicitors are termed either microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs). Since these terms are frequently used interchangeably, we will maintain the broader term MAMP [ 1 ]. MAMPs are detected by pattern recognition receptors (PRRs) that induce various antimicrobial and immune responses to eliminate encroaching infective agents, referred to as MAMP-triggered immunity (MTI) [ 2 , 3 ].…”