Enzymeヽatalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the co﹕ubstrate, biotin﹑henol, into ...
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. This is a preview of ...
Integral membrane protein 2B ITM2B 3 6 1,940,600 Peptidyl-prolyl cis-trans isomerase D PPID 3 5.8 753,450 Retinoic acid-inducible gene 1 protein(RIG-1) DDX58 3 2.5 945,280 MAP7 domain containing 1 MAP7D1 3 2.3 292,250 von Willebrand factor A domain containing 8 VWA8 3 1.2 57,491...
SOPP3-assisted biotinylation activity of APEX2 is proximity dependent, we employed FRB–FKBP interaction system9to control the proximity between APEX2 and SOPP3 via rapamycin induction. The mitochondrial outer membrane (OMM)-localized APEX2 (facing cytosol, signal peptide of OMM protein — TOM20) ...
After 48 h, the supernatant was harvested, filtered through a low-protein binding HT Tuffryn® membrane with 0.45 μm pores (Pall cat. #4184), and stored at −80°C. Lentiviral titer was determined by transducing the cell line of interest plated at clonogenic density with serial dilution...
Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane‐enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques...
However, APEX2 fusion is often deleterious to the protein expression, which seriously hampers its wide utility. This problem is especially compelling when APEX2 is fused to structurally delicate proteins, such as multi﹑ass membrane proteins. In this work, we found that a cysteine‐free single ...
Here, we have performed a comprehensive analysis of the Ras isoform-specific interactome with particular interest in the role of cell surface nanoclustering and plasma membrane organization. We have screened the proteome microenvironment in living mammalian cells via APEX2, an engineered promiscuous ...
G protein-coupled receptors (GPCRs) represent a large family of membrane receptors involved in important physiological functions through regulation of myriad downstream signalling pathways. Many pathways work via heterotrimeric G proteins. In addition to the important role played by Gα subunits, recent...
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 protei