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A Promiscuous Biotin Ligase as a Global Strategy to Interrogate the Protein Interactome of Human ABCC4
Author(s) -
LooiSomoye Remi,
Wijaya Juwina,
Peng Junmin,
Schuetz John
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.695.18
Subject(s) - biotinylation , biology , immunoprecipitation , microbiology and biotechnology , pdz domain , membrane protein , biochemistry , gene , membrane
ATP‐Binding Cassette transporter sub‐family C member 4, ABCC4, is a plasma membrane transporter with the ability to export prostaglandins and cancer chemotherapeutics. In acute myeloid leukemia (AML), ABCC4 interacts with a binding protein through its PDZ motif1. This interaction with a highly expressed PDZ protein, a protein which correlated with worse overall survival for AML patients, affects ABCC4 plasma membrane localization and retention, leading to profound effects on its function and drug resistance. The intense focus on ABCC4's PDZ motif as a locus for protein interaction has left other potential protein interactions that might regulate ABCC4 function largely unexplored. Little is actually known about how ABCC4 traffics to and from the plasma membrane. In the present study, we developed a global approach to not just identify potential interacting proteins, but also determine the proteins required for ABCC4 trafficking and internalization. We used a promiscuous biotin ligase, BioID2, which covalently attaches a biotin moiety to nearby proteins in the presence of biotin. Fusing BioID2 to the N‐terminus of ABCC4 did not alter its glycosylation, plasma membrane localization, or function. Upon addition of biotin, the BioID2‐ABCC4 fusion protein was capable of biotinylating itself as well as ABCC4 proximal and interacting proteins. We then developed an immunoprecipitation and elution protocol and strategy to specifically identify proteins biotinylated by the BioID2‐ABCC4 fusion protein. With successful immunoprecipitation of biotinylated proteins and identification by mass spectrometry, proteins within the vicinity of ABCC4, from its genesis at the endoplasmic reticulum to its final destination at the plasma membrane, will be identified. At the same time, proteins involved in ABCC4 recycling to degradation will also be discovered. Overall, this study will provide unique insights into the mechanisms of ABCC4 trafficking and identify proteins that will allow for modulation of ABCC4 level. This will be beneficial for malignancies associated with high ABCC4 expression, including neuroblastoma, prostate, non‐small cell lung, and endometrial cancers. Support or Funding Information This work was supported by NIH and ALSAC. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .