A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for DNAJC13 in opioid receptor downregulation

ABSTRACT: G protein-coupled receptor (GPCR) activity is critically regulated by trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-receptor interactions. Here we show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). Using our GPCR-APEX2/AUR pipeline, we demonstrate that a gene identified in our screen, DNAJC13, plays a conserved role in the trafficking of activated DOR to the lysosome. To determine how depletion of DNAJC13 changes the local proteome of DOR-positive endosomes in cultured cells, we made cell lines stably expressing DOR-APEX2 or GFP-APEX2 (cytoplasmic). We then used siRNAs to knockdown DNAJC13 from DOR-APEX2 cells and compared these results to a siRNA control. Three independent biological replicates of the proximity labeling reaction were performed follow by purification of the biotinylated proteins, trypsinization, purification of peptides, TMT labeling, and detection and analysis by MS. In addition, this TMT-dataset (18-plex) contains 6 additional samples of from an independent experiment analyzing the proximity labeling of a 2xFYVE-GFP-APEX2 construct, but these samples were not included in further data analysis. We found that the DOR proximal proteome was highly enriched in endosomal proteins compared to the cytoplasmic control, and found 13 proteins significantly changed (FDR<0.1) with DNAJC13 knockdown.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293 Cell

SUBMITTER: Hayden Adoff  

LAB HEAD: Braden Lobingier

PROVIDER: PXD044921 | Pride | 2024-09-03

REPOSITORIES: Pride