Characterization of Sec63-mediated clearance of translocon-occupied marginally hydrophobic TMDs. (A) A cartoon depicting the predicted topology of WRB fused with the C-terminus Venus tag. The red circle in the C-TMD (third TMD) indicates the charged lysine amino acid. The amino acid sequence and free energy prediction for all three TMDs of WRB are shown. The C-TMD has a high free energy (∆Gapp) value because it contains a positively charged lysine residue. (B) The C-TMD of WRB-Venus is mutated to either increase the free energy by introducing charged residues as specified in red color or reduce the free energy by introducing hydrophobic leucine residues as indicated in blue color (Sun and Mariappan, 2020). The free energy of all C-TMD variants was predicted as previously described (Hessa et al., 2007). (C) WT HEK293 or Sec63^−/− cells were transfected with the indicated substrates with varying lengths of amino acids after their C-TMDs. Cells were metabolically labeled for 30 min and immunoprecipitated with anti-HA antibody beads. The immunoprecipitants were analyzed by autoradiography. The star symbol indicates the translocated/glycosylated form. (D) WT HEK293 or Sec63^−/− cells stably expressing the indicated FLAG-tagged Sec63 constructs were lysed, immunoprecipitated with anti-FLAG beads, and analyzed by immunoblotting for indicated antigens. (E) The indicated cell lines were transfected with C-TMDs of substrates carrying the indicated charged amino acid(s). Transfected cells were directly analyzed by immunoblotting with anti-GFP antibodies for substrates. gly. indicates glycosylated forms. ungly. denotes non-translocated/unglycosylated forms with uncleaved SSs. Source data are available for this figure: SourceData FS1.