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. 2022 Dec 2;222(1):e202203070. doi: 10.1083/jcb.202203070

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© 2022 Sun et al.

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Figure 1. — Sec63/BiP are required for releasing marginally hydrophobic TMDs retained at the Sec61 translocon. (A) Diagram illustrating the translocon engaged with the marginally hydrophobic (or weak) C-TMD of WRB-Venus, which is then translocated into the ER lumen and is glycosylated. The C-TMD with sufficient hydrophobicity is correctly inserted into the ER membrane and, thereby, is not glycosylated. The red circle in the C-TMD indicates a charged residue. (B) Cells expressing C-TMDs of substrates bearing indicated charged residues (shown in red color) or hydrophobic residue (marked in blue color) were radiolabeled and analyzed by SDS-PAGE and autoradiography after IP of substrates with anti-GFP antibodies. The molecular weight measurements (kD) are listed beside the immunoblot and all other following immunoblots. gly. indicates glycosylated forms. ungly. denotes non-translocated/unglycosylated forms with uncleaved SSs. (C) The indicated substrates were expressed in chromosomally 2× Strep-tagged Sec61α cells, lysed, pulled down with Strep-Tactin beads, and analyzed by immunoblotting for the indicated antigens. The substrates were immunoblotted with anti-GFP antibodies. (D) Cells co-expressing the indicated versions of BiP and the substrate were analyzed by immunoblotting with anti-BiP or anti-GFP antibodies for substrates. The percent of glycosylation is shown below each panel. (E) WT HEK293, Sec62^−/−, or Sec63^−/− cells expressing the indicated substrates were radiolabeled and analyzed as in B. Immunoblots show the knockout of either Sec62 or Sec63. (F) WT HEK293 or Sec63^−/− cells expressing the indicated substrates were immunoprecipitated with anti-GFP antibodies and analyzed by autoradiography. (G) Transcripts of the indicated substrates possessing or lacking a stop codon were in vitro translated in the RRL, including nothing or CRM. The asterisk symbol denotes tRNA-linked nascent chains. Transcripts with a stop codon produce free nascent chains that are likely ubiquitinated (indicated by black circles) in the cytosol lacking CRM. (H) The indicated transcripts lacking a stop codon were translated in the presence of microsomes derived from WT HEK293 or Sec63^−/− cells. The reactions were treated with PK and analyzed by SDS-PAGE autoradiography. # denotes a background band. (I) Model showing Sec63/BiP-mediated release of a weak TMD retained at the Sec61 translocon. Source data are available for this figure: SourceData F1.