MCFD2 ΔSLQ displays impaired binding to ERGIC-53 due to a structural defect. (A) Amino acid sequence of MCFD2. EF-hand motifs are underlined and the ΔSLQ mutation is boxed. (B) Schematic representation of HA-tagged constructs. The endogenous signal sequence of MCFD2 was replaced by the signal sequence of calreticulin (SS_cal). (C) Cell lysates (5% input) and anti-ERGIC-53 immunoprecipitates (IPαERGIC-53) were probed by Western blotting (WB) using anti-ERGIC-53 and anti-HA antibodies. Because ERGIC-53 antibodies were covalently coupled to immunoprecipitation beads, only the antibody light chain is detectable (*). (D) ERGIC-53 coprecipitated HA-MCFD2 WT and ΔSLQ were quantified in 4 independent experiments and relative values are indicated. (E) Far UV CD spectra of recombinant MCFD2 (—) and MCFD2 ΔSLQ (). Each spectrum shows the average of 4 scans over the range 200 to 250 nm with step size of 0.1 nm and a bandwidth of 1.0 nm.