Fig. 3: CKAP4 is the ER-resident receptor for FKBPL.

a Schematic representation of V5-APEX-FKBPL assay: V5-APEX-FKBPL biotinylates protein of interest ~ 20 nm nearby. The illustration created with BioRender.com is released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. b HEK293T cells were transfected with V5-APEX-FKBPL, left treated or untreated with biotin-phenol for 30 min, and then biotinylated in the presence of H₂O₂ for 1 min. Biotinylated proteins pulled down by Streptavidin-Agarose and WCL were analyzed by immunoblotting. c HEK293T cells were transfected with HA-FKBPL. Immunoprecipitates of anti-HA antibody and WCL were analyzed by immunoblotting. d Immunoprecipitates of anti-CKAP4 antibody and WCL of HEK293T cells were analyzed by immunoblotting. e Domain mapping of FKBPL binding to CKAP4. FKBPL WT and deletion mutants (left). The interaction between CKAP4-mRFP and GFP-FKBPL is noted. +, interaction; −, no interaction (right). f HEK293T cells were co-transfected with CKAP4-mRFP and EGFP-C1 (-) or the indicated GFP-FKBPL constructs. Immunoprecipitates of anti-mRFP antibody and WCL were analyzed by immunoblotting. g Domain mapping of CKAP4 binding to FKBPL. CKAP4 WT and deletion mutants (left). The interaction between HA-FKBPL and CKAP4-mRFP is noted. +, interaction; −, no interaction (right). h HEK293T cells were co-transfected with HA-FKBPL and EGFP-N1 (-) or the indicated CKAP4-GFP constructs. Immunoprecipitates of anti-HA antibody and WCL were analyzed by immunoblotting. Arrow, CKAP4 deletion (Δ130-602); asterisk, non-specific. i Protein structure of human FKBPL predicted by Alphafold. Representing amino acid numbers are indicated. The magenta oval covers the CKAP4-interacting region (amino acids 86–202); the Red oval covers LIR motifs (₁₄₁WTEL₁₄₄ and ₁₅₆WGEL₁₅₉); the Green oval covers FKBPL self-interacting region (amino acids 203–315). j Membrane fractionation with continuous iodixanol gradient was performed using cell extract of HeLa/WT or CKAP4 KO cells. WCL and 12 fractions collected from lighter to heavier ones (Fractions 1 to 12) were analyzed by immunoblotting.