The human proton-coupled folate transporter (PCFT) was stably expressed as a fu-sion protein with a BioID2-HA in the C-terminal position (PCFT-BioID2). The uncom-plxed biotin transferase was used as a control to identify proteins that are biotinylated randomly due to sheer abundance and/or an unnatural affinity to the BioID2 ligase. HeLa PCFT-BioID2 cells expressed the PCFT fusion protein which localized to the plasma mem-brane and exhibited robust transport typical of PCFT. HeLa PCFT-BioID2 and BioID2 cells were cultured in complete DMEM (biotin-free), with and without biotin (50 μM; 16 h) then biotinylated proteins were selected on streptavidin beads.