Kanzler CR et al. (2022), TurboID functions as an efficient biotin ligase...

XB-ART-59407

Dev Biol 2022 Dec 01;492:133-138. doi: 10.1016/j.ydbio.2022.10.005.

Show Gene links Show Anatomy links

TurboID functions as an efficient biotin ligase for BioID applications in Xenopus embryos.

Kanzler CR , Donohue M , Dowdle ME , Sheets MD .

???displayArticle.abstract???
BioID is a proximity labeling strategy whose goal is to identify in vivo protein-protein interactions. The central components of this strategy are modified biotin ligase enzymes that promiscuously add biotin groups to proteins in close proximity. The transferred biotin group provides a powerful tag for purification and thus identification of interacting proteins. While a variety of modified biotin ligases were created for BioID, the original enzymes were inefficient, required long incubation times, and high intracellular biotin concentrations for protein labeling. These limitations hinder the application of BioID in contexts such as developing embryos where processes such as cell division and cell fate decisions occur rapidly. Recently, a new biotin ligase called TurboID was developed that addressed many of the deficiencies of previous enzymes. In this paper we compare TurboID to the BioID2 biotin ligase in developing Xenopus embryos. We find that the TurboID enzyme has several advantages over the BioID2 enzyme. TurboID labels proteins efficiently without the addition of additional biotin and occurs at a range of temperatures compatible with the culturing of Xenopus embryos. Biotinylation events occurred rapidly and were limited by TurboID expression and not its activity. Thus, TurboID is an efficient tool for BioID applications in Xenopus embryos and its use should facilitate the identification of interacting proteins in specific networks and complexes during Xenopus development.

???displayArticle.pubmedLink??? 36270327
???displayArticle.pmcLink??? PMC9643680
???displayArticle.link??? Dev Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: bicc1
GO keywords: enzyme activator activity [+]

???attribute.lit??? ???displayArticles.show???

	Fig. 1. TurboID is an efficient biotin ligase in Xenopus embryos. (A) Diagrams of HA-tagged TurboID and BioID2. (B) Assay for biotin ligase mediated-biotinylation. Two-cell Xenopus embryos were injected with mRNA encoding either HA-tagged BioID2 or HA-tagged TurboID with biotin supplemented in the culture media at indicated concentrations. When embryos reached stage 7, protein was extracted for analysis of biotinylation. (C) Immunoblot analysis with an anti-HA antibody was used to monitor the expression of injected mRNA (Lanes 1â3, top panel). Streptavidin-HRP assayed self-biotinylation of each enzyme as a readout of enzyme activity (Lanes 1â3, middle panel). Blots were stripped and re-probed with actin to assess sample loading (Lanes 1â3, bottom panel). Open triangles indicate where biotinylation of BioID2 is to be expected. Figure is representative of three biological replicates.
	Fig. 2. TurboID requires no exogenous biotin. Experiments were performed as in Fig. 1A with the same constructs used in 1B. Embryos were cultured with 0, 100, or 500 âÎ¼M supplemental biotin added to culture media. Immunoblot analysis with an anti-HA antibody was used to monitor the expression of injected mRNA (Lanes 1â7). Streptavidin-HRP assayed self-biotinylation of each enzyme as a readout of enzyme activity (Lanes 8â14). Blots were stripped and re-probed with anti-actin antibody to assess sample loading (Lanes 15â21). Open triangles indicate where biotinylation of BioID2 is to be expected. Figure is representative of three biological replicates.
	Fig. 3. Biotinylation by TurboID can occur at multiple temperatures in Xenopus embryos. Experiments were performed as in Fig. 1A with the same mRNAs as in 1B. Embryos were cultured at 13.6, 18, or 24C before harvesting proteins for analysis. Immunoblot analysis with an anti-HA antibody was used to monitor the expression of injected mRNAs (Lanes 1â7). Streptavidin-HRP assayed self-biotinylation of each enzyme as a readout of enzyme activity (Lanes 8â14). Blots were stripped and re-probed with anti-actin antibody to assess sample loading (Lanes 15â21). Open triangles indicate where biotinylation of BioID2 is to be expected. Figure is representative of three biological replicates.
	Fig. 4. Biotinylation by TurboID occurs rapidly in Xenopus embryos. Experiments were performed as in Fig. 1A with the same mRNAs as in 1B. Embryos were allowed to develop to stage 4, 7, or 11 before harvesting. As in previous figures, immunoblot analysis with an anti-HA antibody was used to monitor the expression of injected constructs (Lanes 1â7). Streptavidin-HRP assayed self-biotinylation of each enzyme as an indication of enzyme activity (Lanes 8â14). Blots were stripped and re-probed with anti-actin antibodies to assess sample loading (Lanes 15â21). Open triangles indicate where biotinylation of BioID2 is to be expected. Figure is representative of three biological replicates.
	Fig. 5. Protein-protein interactions are efficiently identified with TurboID fusion proteins. (A) Assay for pulldown of biotinylated proteins post proximity-labeling with Bicc1-biotin ligase fusion proteins. Two-cell Xenopus embryos were injected with mRNA encoding either HA-Bicc1-BioID2 or HA-Bicc1-TurboID fusion proteins along with HA-Bicc1. When embryos reached stage 7, protein was extracted for streptavidin purification of biotinylated proteins. (B) Diagrams of HA-Bicc1-BioID2 or HA-Bicc1-TurboID fusion proteins. (C) Immunoblot analysis of streptavidin-purified proteins with an anti-HA antibody was used to observe pulldown, and hence biotinylation, of HA-Bicc1, as well as self-biotinylation of the fusion proteins (Lanes 2 & 4). Blots were stripped and re-probed with anti-tubulin and anti-GAPDH antibodies as a specificity controls. Figure is representative of three biological replicates.

References [+] :

Ahmed, Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system. 2014, Pubmed