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. 2024 Nov 12;224(1):e202406103. doi: 10.1083/jcb.202406103

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© 2024 MRC Laboratory of Molecular Biology

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S4. — Co-translational and SP-dependent interaction of clients with SURF4. (A) Doxycycline induction test to optimize concentration for FLAG-SURF4 expression. A western blot of cell lysates, probed with an antibody for endogenous SURF4 is shown. (B) Proteinase K (PK) protection assay for various Cab45 constructs, examining the effects of indicated mutations at and near the SP cleavage, ER-ESCAPE and N-glycosylation sites with respect to glycosylation and SP cleavage. The size of PK-protected (i.e., ER membrane-enclosed) Cab45-40N* band is the same as that of a glycosylation mutant Cab45-N40Q (and Cab45-EEE [ER-ESCAPE mutation] as well as Cab45-EEE-N40Q), lower than that of Cab45 WT (no amber) and much lower than Cab45-SP-N40Q, whereby SP cleavage is disrupted (as per schematic below). This indicates that Bpa incorporation at position 40 (as well as ER-ESCAPE mutation into EEE) does not disrupt SP cleavage but instead prevents N-glycosylation. The higher Mw form of Cab45-40N* seen in Fig. 4 B IPs is therefore likely SP-uncleaved form that remains in close proximity to the ER membrane and therefore is IPed together. The gel was ran until 26 kDa marker ran out to have better separation of the different Cab45 intermediates. (C) Proteinase K (PK) protection assay of various Cab45 constructs examining the effects of indicated mutations at and near the SP cleavage, ER-ESCAPE and N-glycosylation sites with respect to membrane glycosylation and SP cleavage. The gel was ran until 17 kDa marker ran out to have better separation of the different Cab45 intermediates. (D) Site-specific Cab45-HA photo-crosslinking experiment showing Cab45-SURF4 direct interaction is ER-ESCAPE-dependent and still occurs with the N-glycan present. Semi-permeabilized cells were treated with S7 nuclease to reduce doxycycline-induced FLAG-SURF4 background. (E) Samples as in C, but Cab45-HA and FLAG-SURF4 IPs were performed from UV-crosslinked semi-permeabilized cells. Green arrows indicate Cab45-SURF4 cross-links. (F) n = 2. (G) Cab45 C-terminal truncations photo-crosslinked to TRAPα with Bpa placed at the N-glycosylation site of Cab45 (N40*). (H) NUCB1 C-terminal truncations photo-crosslinked to FLAG-SURF4 immunoprecipitated from UV-crosslinked semi-permeabilized cells. Source data are available for this figure: SourceData FS4.