Abstract
Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.
Highlights
Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes
We constructed sets of truncations for optimizing the APEX2 complementary pairs which fused to FKBP12-rapamycin binding (FRB) and FKBP12
Because the APEX2 enzyme can catalyze the H2O2-dependent polymerization of DAB, leading to localized precipitation[19,20], it is easy to visualize the complementation by common light microscopy (Fig. 1A)
Summary
Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. An ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells. Live cells, has potential for direct labeling but is not suitable for interactions within cells, a consequence of spontaneous reconstitution in the endoplasmic reticulum (ER)[16]
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