| 摘要 | The communication between cells and the communication between cellular organelles are often be controlled by the interaction of membrane proteins.Despite of many methods to detect protein-protein interactions(PPIs),there are still challenges in detecting membrane PPIs.Firstly,transient and weak PPIs are mostly associated with membrane receptor-mediated signaling pathways.Secondly,mass spectrometry-based membrane protein interaction studies have not been widely successful due to the poor solubility of membrane proteins or the loss of PPIs in sample preparation.Recent years,protein proximity tagging methods,such as APEX and BioID,have been applied in membrane protein study and showed great results.Here we developed another method to specifically tag the interacting proteins of MPOI(membrane protein of interest) in cells.This approach can transform transient and weak interactions into covalent binding and the tagged proteins can be enriched by affinity purification under denaturing conditions for mass spectrometry-based identification.We applied this approach to CD28,a critical co-stimulatory receptor for T lymphocyte activation.Three known CD28 binding partners as well as multiple potential interacting proteins were identified.In addition,this system is proved to be efficient to track cell-cell interactions.Finally,we show that this method can be used to identify the interaction between a cell surface receptor and its ligand.In general,we designed a powerful tool to uncover the weak and transient membrane PPIs in cells,which will have a general application in the study of membrane proteins. |
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