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, 12 (1), 51-4

Directed Evolution of APEX2 for Electron Microscopy and Proximity Labeling

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Directed Evolution of APEX2 for Electron Microscopy and Proximity Labeling

Stephanie S Lam et al. Nat Methods.

Abstract

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

Figures

Figure 1
Figure 1
Yeast display evolution of APEX2 for electron microscopy (EM) and proteomics applications. (A) Structure of wild-type soybean ascorbate peroxidase (APX) with mutations present in APEX, APEX2, and VPGAPEX indicated. New mutations discovered in this study are highlighted yellow. The active site is magnified to show the heme cofactor, aromatic substrate (salicylhydroxamic acid, brown) binding site, and A134 position (yellow) that is mutated to proline in APEX2. From PDB ID 1 V0H. (B) Scheme showing how APEX or APEX2 can be used as a reporter to generate contrast for EM. (C) Scheme showing how APEX or APEX2 can be used for proteomic tagging in living cells., Blue B, biotin. (D) Labeling and selection scheme used to evolve APEX2 by yeast display. Biotin-phenol was added to a dilute yeast suspension for 1 minute to allow cells displaying highly active APEX variants to promiscuously biotinylate themselves. Minimal inter-cellular labeling was observed under these conditions. Biotinylation sites (blue) were stained with fluorescent streptavidin-phycoerythrin (red), and APEX expression level was quantified via anti-myc antibody staining (yellow). Two-dimensional fluorescence activated cell sorting (FACS) was used to enrich for cells displaying the highest activity/expression ratio (i.e., streptavidin/myc ratio). (E) FACS plot showing the initial APEX mutant library, and the sorting gate used in the first round of selection (red polygon). Single trial, 10,000 cells shown. FACS analyses of enriched populations and individual clones shown in Supplementary Figure 2.
Figure 2
Figure 2
APEX2 has improved cellular activity and sensitivity for proteomic tagging and electron microscopy. (A) HEK cells expressing the indicated APEX variant were labeled with biotin-phenol for 1 minute before fixation and staining with streptavidin-AlexaFluor568 (red) to visualize biotinylation sites, and anti-Flag antibody (cyan) to visualize APEX expression. Arrowheads, cells with low APEX2 expression and strong biotinylation. Asterisks, cells with high APEX expression and low biotinylation. DIC, differential interference contrast. Images representative of 25 fields of view. Scale bars, 50 μm. (B) Quantitation of experiment shown in (A). >50 single cells were analyzed across >16 fields of view for each APEX variant. Mean streptavidin intensities are plotted ± 1 s. d. (C) Comparison of proximity dependent biotinylation by APEX2 and APEX. Live HEK cells expressing APEX or APEX2 targeted to the endoplasmic reticulum (ER) membrane (ERM) or outer mitochondrial membrane (OMM) were labeled with biotin-phenol as in (A). After cell lysis, biotinylated proteins were enriched using streptavidin beads and blotted for the endogenous ER and mitochondrial proteins shown. (D) Comparison of APEX2 and APEX for EM imaging of HEK cells expressing plasma membrane-targeted constructs. Arrowheads point to plasma membrane. Images representative of >3 fields of view. Scale bars, 500 nm. For (C) and (D), controls showed that APEX and APEX2 construct pairs were expressed at similar levels (data not shown). (E) Purified peroxidases were incubated with 1.4 mM or 0.5 mM guaiacol (a model aromatic substrate, ) to mimic EM and proteomic tagging conditions, respectively. Initial rates (Vo) were measured for a range of H2O2 concentrations. Each plot is representative of 2-5 trials.
Figure 2
Figure 2
APEX2 has improved cellular activity and sensitivity for proteomic tagging and electron microscopy. (A) HEK cells expressing the indicated APEX variant were labeled with biotin-phenol for 1 minute before fixation and staining with streptavidin-AlexaFluor568 (red) to visualize biotinylation sites, and anti-Flag antibody (cyan) to visualize APEX expression. Arrowheads, cells with low APEX2 expression and strong biotinylation. Asterisks, cells with high APEX expression and low biotinylation. DIC, differential interference contrast. Images representative of 25 fields of view. Scale bars, 50 μm. (B) Quantitation of experiment shown in (A). >50 single cells were analyzed across >16 fields of view for each APEX variant. Mean streptavidin intensities are plotted ± 1 s. d. (C) Comparison of proximity dependent biotinylation by APEX2 and APEX. Live HEK cells expressing APEX or APEX2 targeted to the endoplasmic reticulum (ER) membrane (ERM) or outer mitochondrial membrane (OMM) were labeled with biotin-phenol as in (A). After cell lysis, biotinylated proteins were enriched using streptavidin beads and blotted for the endogenous ER and mitochondrial proteins shown. (D) Comparison of APEX2 and APEX for EM imaging of HEK cells expressing plasma membrane-targeted constructs. Arrowheads point to plasma membrane. Images representative of >3 fields of view. Scale bars, 500 nm. For (C) and (D), controls showed that APEX and APEX2 construct pairs were expressed at similar levels (data not shown). (E) Purified peroxidases were incubated with 1.4 mM or 0.5 mM guaiacol (a model aromatic substrate, ) to mimic EM and proteomic tagging conditions, respectively. Initial rates (Vo) were measured for a range of H2O2 concentrations. Each plot is representative of 2-5 trials.
Figure 3
Figure 3
EM analysis of MICU1 using APEX2 supports intermembrane space (IMS) localization. (A) Scheme showing conflicting models that localize MICU1 to the IMS or matrix space, of mitochondria. Due to porins in the outer mitochondrial membrane (OMM), the IMS is permeable to cytosolic Ca2+, in contrast to the matrix. (B) Western blot characterization of MICU1-APEX2 stable HEK cells. Endogenous MICU1 was knocked out (MICU1 KO) and replaced with either MICU1-APEX2 or MICU1-Flag. Blotting was performed for MICU1 or succinyl dehydrogenase subunit B (SDHB), another IMM-localized protein loading control. (C) Representative traces (n=3) showing mitochondrial calcium uptake in response to a CaCl2 pulse resulting in ∼1 μM [Ca2+]free by monitoring Fluo4 fluorescence. Color coding explained in (D). (D) Graph reporting the linear fits of calcium uptake data from (C) between 50-60 sec after CaCl2 addition. Values are normalized to that of MICU1 KO (n=3). Error bars, ± 1 s.d. (E) EM images of HEK cells stably expressing MICU1-APEX2 (characterized in (B)-(C)). Two fields of view are shown. Dark stain generated by APEX2 is observed exclusively in the IMS, and not matrix space of mitochondria. Arrowheads point to wider cristae regions where dark stain is associated with the IMM rather than filling the IMS. Mitochondria of untransfected HEK cells processed under identical conditions are shown at right. Images representative of > 9 fields of view. Scale bars, 500 nm.

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