Figure 4.
Sec63 substrates are co-translationally translocated with an initial pause. (A) Scheme depicting the PK treatment protocol for the co- or post-translational protein translocation assay. (B) For the co-translational protein translocation, transcripts encoding AGAL-FLAG were in vitro translated in RRL, including either buffer or CRM. After translation, samples were digested with PK and analyzed by autoradiography after IP with an anti-FLAG antibody. For the post-translational translocation, transcripts encoding AGAL-FLAG were translated and centrifuged to remove ribosomes. The supernatant was incubated with CRM, digested with PK, and analyzed after IP with an anti-FLAG antibody. (C) AGAL or Prl-AGAL transcripts lacking a stop codon were in vitro translated in RRL, including either buffer or CRM. The reactions were treated with PK and analyzed as in B. (D) The indicated lengths of AGAL RNCs were produced in RRL, including either buffer or CRM, digested with PK, treated with RNase A to remove tRNA, and analyzed by autoradiography. The percent of protease-protected fragments (PF) is shown below each panel. The star symbol indicates the distortion of AGAL-130 nascent chain caused by the co-migration with hemoglobin from RRL. (E and F) Nascent chains of the indicated lengths of Prl-AGAL or 3L-AGAL were produced and analyzed as in D. (G) Nascent chains of AGAL-220, Prl-AGAL-220, or 3L-AGAL-220 were produced in the absence or presence of CRM. Translation reactions were denatured, treated with or without Endo H, and analyzed by autoradiography. (H) The membrane-targeted nascent chains of AGAL-100 or Prl-AGAL-100 were isolated by centrifugation and treated with bismaleimidohexane (BMH) crosslinker. An aliquot was directly analyzed (input), while the remainder was denatured and immunoprecipitated with the indicated antibodies. Anti-GFP antibodies were used as a control. The nascent chain (NC) crosslinked adducts are shown by “NC100 ×.” (I) Transcripts of AGAL-220 or 3L-AGAL-220 lacking a stop codon were translated in the presence of either rough microsomes (RM) derived from WT HEK293 cells or Sec63−/− cells and digested with PK before analyzing by autoradiography. Note that protease-protected fragments mostly disappeared in samples containing 1% Triton X-100, suggesting that they are translocated into the ER. Source data are available for this figure: SourceData F4.