Abstract
Proximity-dependent biotin identification (BioID), which detects physiologically relevant proteins based on the proximity-dependent biotinylation process, has been successfully used in different organisms. In this report, we established the BioID system in rice protoplasts. Biotin ligase BirAG was obtained by removing a cryptic intron site in the BirA∗ gene when expressed in rice protoplasts. We found that protein biotinylation in rice protoplasts increased with increased expression levels of BirAG. The biotinylation effects can also be achieved by exogenous supplementation of high concentrations of biotin and long incubation time with protoplasts. By using this system, multiple proteins were identified that associated with and/or were proximate to OsFD2 in vivo. Our results suggest that BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment in plant cell.
Highlights
Proteins perform a vast array of functions within living organisms, including catalyzing enzymatical reactions, DNA replication, stimuli responding, and molecule transporting from one location to another
We expressed BirAG in rice protoplasts, and observed a single transcript indicating that the cryptic sites for intron splicing in BirA∗ has been removed (Figure 1D)
The parameters influencing the biotin ligase functioning in rice cell such as gene expression level, biotin concentration and protoplast incubation time were studied, which forms the basis of using biotin identification (BioID) system in plant cell
Summary
Proteins perform a vast array of functions within living organisms, including catalyzing enzymatical reactions, DNA replication, stimuli responding, and molecule transporting from one location to another. Most proteins carry out their functions together with other proteins in a protein complex. Protein–protein interactions are a hallmark of all essential cellular processes (Ellis, 2001; Tabaka et al, 2014). Detection of protein–protein interaction in vivo is an essential part to understand the function of individual proteins at molecular level. Many methods have been developed and used to screen and identify the interacting proteins, such as the yeast-two hybrid system (Y2H), the co-immunoprecipitation (Co-IP) and the bimolecular fluorescence complementation (BiFC), etc. Some methods can be merely used in vitro, which may not reflect the actual interaction inside cells, or suffer the loss of candidate proteins because of protein insolubility or transient or weak protein interactions (Roux et al, 2012)
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