Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: Blots of lysates of HeLa cells transiently transfected with the indicated constructs and incubated with 50 μM biotin for 10 min. Biotinylation was analyzed using IRDye680-labeled streptavidin and expression levels of the fusion proteins with antibodies against the Myc tag as indicated. a Comparison microID, ultraID-4 and -5, and TurboID. b Comparison microID, microID L41P-only, microID R40G/L41P (=ultraID), microID S24C/R40G/L41P (=ultraID-4 with K169R reverted to WT) and TurboID.

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Transfection, Construct, Incubation, Labeling, Expressing, Comparison

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: a Blots of lysates of S. cerevisiae strains expressing the indicated Asc1 fusion proteins and incubated or not (−) with 10 μM biotin for the indicated times. The fusion proteins were expressed from plasmids in a Δ asc1 strain, and the ASC1 wild-type strain carrying the empty vector served as a control. Biotinylation was analyzed using HRP-labeled streptavidin and expression levels of the fusion proteins with antibodies against Asc1 as indicated. b Blots of lysates of E. coli cells transformed with expression plasmids for ultraID, microID, TurboID, or the control plasmid. The cells had been incubated (+) or not (−) with biotin for 16 h. The arrowhead indicated the expected size for bacterial GAPDH, and the asterisk (*) indicates a non-specific cross-reactivity that was used as a loading control.

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Expressing, Incubation, Plasmid Preparation, Control, Labeling, Transformation Assay

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: a Experimental set-up for the side-by-side comparison of overnight labeling with BioID vs. 10 min labeling with ultraID or TurboID. b Venn diagram displaying the proteomic dataset sizes and overlap of the significant hits from a , the gene names of the dataset overlap are specified. c Relative abundance, assessed with iBAQ values, of endogenous biotinylated carboxylases and main interacting partners of AGO2 (TNRC6A & B) and RAB11A (RAB11FIP1 & 5), obtained from HeLa cells expressing TurboID-Ago2 and ultraID-Ago2 (left) or TurboID-Rab11and ultraID-Rab11 (right) after streptavidin pulldowns with no biotin addition. Bars are mean values, n = 3 (Ago2 cell lines) or 4 (Rab11 cell lines).

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Comparison, Labeling, Expressing

Journal: Communications Biology

Article Title: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

doi: 10.1038/s42003-022-03604-5

Figure Lengend Snippet: a Blots of lysates of P19 cells stably expressing ultraID-Ago2, of P19 cells knock-out for endogenous γ1-COP and rescued by γ1-COP-ultraID, and of P19 cells knock-out for γ2-COP and rescued by γ2-COP-ultraID lines. The fusion proteins were detected with antibodies against γ1- and γ2-COP and biotinylation with IRDye680-labeled streptavidin. b Experimental set-up for the determination of the membrane-associated interactome of γ1-COP and γ2-COP. c Volcano plots of the proteins identified by LC-MS/MS with the mock vs. BFA samples. Significant hits ( p value <0.01 and log 2 fold change >2 in this analysis, and p value <0.01 and log 2 fold change >2 in the γ-COP vs. Ago2 comparison under mock conditions), are indicated with gene names in red. d Proteins identified as membrane interactors of γ1-COP and γ2-COP (see text).

Article Snippet: Plasmids for bacterial and mammalian expression of microID and ultraID are available at Addgene (Plasmid IDs 172878–172881).

Techniques: Stable Transfection, Expressing, Knock-Out, Labeling, Membrane, Liquid Chromatography with Mass Spectroscopy, Comparison