Figure 5. XBP1u nascent chains interact with the Sec61 translocon, but inefficiently insert into the ER membrane.

(A) The membrane-targeted RNCs of XBP1u or XBP1u-TR were isolated by centrifugation and treated with BMH crosslinker. An aliquot was directly analyzed (input, 4% loading), while the remainder was IP with the indicated antibodies. Anti-GFP antibodies were used as a control. The XBP1u crosslinked adducts are indicated by ‘XBP x’. * indicates an unidentified crosslinked product. (B) The ER membrane targeted RNCs of XBP1u variants were subjected to a proteinase K (PK) accessibility assay in the presence of the indicated salt concentrations and salt plus puromycin (pur.). * indicates the inclusion of a detergent in the reaction. FL indicates full-length versions of XBP1u. Band 1 indicates protease-protected fragments of either ribosome translocon protected fragments or protected fragments after insertion into the membrane (lane 9). Band 2 indicates fragments protected by ribosomes. (C) The indicated versions of XBP1u transcripts containing a glycan acceptor site at the C-terminus were translated in vitro in the presence of RM. The reactions were stopped at the indicated time points by directly mixing with the sample buffer and analyzed by autoradiography. gXBP1u denotes the glycosylated form. (D) Cell lysates from HEK 293 cells expressing the indicated FLAG tagged XBP1u versions containing a glycan acceptor site were treated with endoglycosidase H (EndoH) or peptide-N-glycosidase F (PNGase) and analyzed by IB with FLAG.

DOI: http://dx.doi.org/10.7554/eLife.07426.010

Figure 5—figure supplement 1. Insertion assays with XBP1u and its variants.

For the co-translational protein insertion, versions of XBP1 transcripts containing a glycan acceptor site were translated in vitro in the presence of liposomes or RM. The reactions were stopped after 30 min by directly mixing with the endoglycosidase H (EndoH) denaturation buffer and digested with Endo H before analyzing by autoradiography. gXBP1u denotes the glycosylated form of XBP1u. For the post-translational protein insertion, the indicated XBP1u transcripts were initially translated in the absence of RM and the nascent chains were released by the puromycin treatment. The post ribosomal supernatant was used for the insertion assay.

DOI: http://dx.doi.org/10.7554/eLife.07426.011