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, 27 (8), 1188-96

An Improved Smaller Biotin Ligase for BioID Proximity Labeling

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An Improved Smaller Biotin Ligase for BioID Proximity Labeling

Dae In Kim et al. Mol Biol Cell.

Abstract

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.

Figures

FIGURE 1:
FIGURE 1:
Promiscuous biotinylation by BioID2. (A) The dimensions of E. coli (left; PDB ID 1BIA) and A. aeolicus (right; PDB ID 2EAY) biotin ligases based on prior structural analyses. The catalytic (yellow), ATP-binding (green), and DNA-binding (red) domains. (B) BioID and BioID2 were fused with LaA and expressed in HEK293 cells. Fusion proteins were detected by specific antibodies against BioID or BioID2, respectively (red). Biotinylated proteins were labeled with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Scale bar, 10 μm. (C) Proteins biotinylated by BioID-LaA, BioID2-LaA, BioID-only, or BioID2-only were detected with HRP-conjugated streptavidin after SDS–PAGE separation. Expression of either promiscuous ligase leads to biotinylation of endogenous proteins (left). Fusion proteins were detected with anti-myc antibodies (right). (D) BioID-human Sun2 or BioID2-human Sun2 were transiently expressed in NIH3T3 cells. Fusion proteins were detected using an anti-Sun2 antibody incapable of detecting murine Sun2. Scale bar, 10 μm. (E) The NE/ER ratio of the mean intensity of BioID-human Sun2 or BioID2-human Sun2 detected with anti-human Sun2. Values are mean ± SEM. We measured 48 nuclei/condition.
FIGURE 2:
FIGURE 2:
BioID2 requires less biotin than does BioID for promiscuous biotinylation. (A) In vitro biotinylation at variable biotin concentrations was performed using purified BioID (left) and BioID2 (right). Values are mean ±SEM. ****p < 0.0001 and **p < 0.01 as compared with the 50 μM concentration. Each group consisted of three replicates. (B) Cellular biotinylation at variable biotin concentrations was analyzed using cells stably expressing BioID-LaA (left) and BioID2-LaA (right) in culture media supplemented with 10% fetal bovine serum. Biotinylation was measured after incubation with the indicated concentration of biotin for 16 h. (C) Cellular biotinylation at variable biotin concentration was analyzed using cells stably expressing BioID-LaA (left) and BioID2-LaA (right). Biotin-depleted medium was used to inhibit basal biotinylation. Biotinylation by BioID-LaA or BioID2-LaA was analyzed after incubation with the indicated concentration of biotin for 16 h.
FIGURE 3:
FIGURE 3:
Application of BioID2 to the human Nup107–Nup160 complex. (A) Expression of Nup43-BioID or Nup43-BioID2 biotinylated endogenous proteins at the NPC. The NPCs were labeled with an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). To observe more clearly the NPCs, confocal images were taken at the surface of the NE. Scale bar, 10 μm. (B) Proteins biotinylated by Nup43-BioID and Nup43-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were detected with anti-HA antibody (middle). BioID- or BioID2-only controls were detected by an anti-myc antibody (bottom). Asterisk indicates predicted migration of Nup96 and Nup107. (C) Model of the Nup107–Nup160 complex based on the previous literature and resolved structures (Hoelz et al., 2011; Bui et al., 2013). Candidates identified by both Nup43-BioID (middle) and Nup43-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. Modified from Kim et al. (2014). (D) The full range of NPC candidates, with those identified by both Nup43-BioID (left) and Nup43-BioID2 (right) labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue. Modified from Kim et al. (2014).
FIGURE 4:
FIGURE 4:
An extended flexible linker increases the number of candidates detected by Nup43-BioID2. (A) Linear model of Nup43-BioID2 and Nup43-Linker-BioID2 fusion proteins. An extended flexible linker consisting of 13 repeats of GGGGS predicted to provide an ∼25-nm extension was inserted between the Nup43 bait and BioID2 ligase. (B) Expression of Nup43-BioID2 or Nup43-Linker-BioID2 led to biotinylation of endogenous proteins at the NPC. NPCs were labeled using an anti-Nup153 antibody (red). Biotinylated proteins were detected with streptavidin (green). DNA was labeled with Hoechst dye 33258 (blue). Images were taken at the surface of the NE by confocal microscopy. Scale bar, 10 μm. (C) Proteins biotinylated by Nup43-BioID2 and Nup43-Linker-BioID2 were detected with HRP-conjugated streptavidin (top). Fusion proteins were labeled with anti-HA antibody (bottom). (D) Nup107–Nup160 complex candidates identified by both Nup43-BioID2 (middle) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with blue. (E) For the entire NPC, Nups identified by both Nup43-BioID2 (left) and Nup43-Linker-BioID2 (right) are labeled gray. Uniquely detected candidates are colored in green, and fusion proteins are indicated with patterned blue.

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