Fig 2: SEC23B p.V594G can exist in the nucleolus independent of canonical COPII localization and function. (A) Immunofluorescence staining of Nthy-SEC23B-V594G cells with anti-SEC24C antibody and DAPI, followed by imaging with confocal microscopy. Scale bars represent 25 µm. (B) Multiplex analysis of secretome analytes extracted from supernatants derived from Nthy-EGFP, Nthy-SEC23B-WT and Nthy-SEC23B-V594G thyroid cells. Data represent analytes with concentrations ≥100 pg/ml. All other analytes are displayed in Supplementary Material, Table S1. Values represent two technical replicates for which data represent mean values ± SEM. **P < 0.01 and *P < 0.05 (two-sided Student’s t test).

Fig 3: TMED2 and TMED10 do not participate in Cab45 or NUCB1 secretion. (A) Close-up view of the co-essentiality Browser’s neighborhood “ER-to-Golgi body transport”. Wainberg et al. (2021) visualized this co-essentiality by plotting strongly co-essential genes together, as determined by their generalized least squares method. Overlapping Depmap co-dependencies are outlined in grey and overlapping DepMap+OpenCell interactions have a grey grid added. (B) HEK-293TREx cells were treated with the indicated Silencer Select siRNAs for the indicated number of days, alongside a negative siRNA control. For each TMED treatment, lysates were blotted for both TMED2 and TMED10. Actin served as loading control. Conditions in bold were chosen for pulse-chase and NanoBiT experiments. (C) Radiolabeled pulse-chase of Cab45 and NUCB1. HEK-293TREx cells were transfected with the indicated siRNAs, then 24 h later transfected with a plasmid expressing Cab45-HA or NUCB1-HA, which were detected by pulse-chase and immunoprecipitation the following day. Protein secretion was quantified from autoradiographs following SDS-PAGE. Each pulse-chase experiment is representative of three biological replicates and is quantified in D. (E) SURF4/SEC24C double KO cells were transfected with the indicated siRNAs, then 24 h later were cotransfected with SmBiT-SEC24C and LgBiT-SURF4 NanoBiT constructs. Luciferase luminescence values were measured and normalized to WT. Triangles represent mean and error bars represent SD. (F) DSP-crosslinking co-immunoprecipitation of endogenous Cab45 from cells expressing SP-HA-TMED10 WT, coiled-coil, and GOLD domain deletion mutants. Statistical tests were one-way ANOVA with Dunnett’s correction for multiple testing. Data distribution was assumed to be normal but this was not formally tested. ns = not significant, **** = P value <0.0001. For each NanoBiT experiment, six technical replicates were used in each of the three independent biological replicates, as indicated by differential coloring within superplots. Triangles represent the mean and error bars represent SD. Source data are available for this figure: SourceData FS3.

Fig 4: Cargo secretion in SEC24C KO, NanoBiT mutant stability and SURF4 mutagenesis. (A) Radiolabeled pulse-chase experiment testing Cab45 secretion in WT and SEC24 KO cells. Transiently transfected HA-tagged Cab45 was immunoprecipitated from media and lysates at indicated time points after [35S]-Met/Cys addition and detected by SDS-PAGE and phosphorimage analysis. n = 1. (B) Stability of indicated SEC24 constructs was tested in appropriate KO cells by immunoblotting for steady-state protein levels using the antibodies indicated. β-actin served as loading control. (C) SURF4 topology prediction based on trRosetta, TOPCONS, and other topology prediction algorithms (see Materials and methods), with conservation and disease variants mapped from gnomAD. Based on this prediction, mutants in cytosolic or lumenal regions were generated. Those labeled in green indicate defects in Cab45 secretion but stable expression of the mutant. (D) Immunoblots of steady-state FLAG-SURF4 and secreted Cab45 upon transient expression of FLAG-SURF4 WT and mutant constructs in HEK-293TREx SURF4 KO. Actin serves as a loading control for lysates. Mutants in green had reduced or no Cab45 secretion rescue in the media. (E) Stability of indicated SURF4 constructs was tested in appropriate KO cells by immunoblotting for steady-state protein levels using the antibodies indicated. β-actin served as a loading control. Source data are available for this figure: SourceData FS1.

Fig 5: Mutagenesis reveals complexity of SURF4 interactions with SEC24 paralogs. (A) AlphaFold2 model of SURF4, highlighting cytosolic regions important for SURF4-dependent cargo secretion identified by mutagenesis. (B) Luminescence values were measured upon co-expression of SmBiT-SEC24C or SmBiT-SEC24A with the indicated LgBiT-SURF4 mutants, normalized to WT values. (C) Crystal structure of SEC24A (PDB ID: 5VNO) showing the three binding sites tested in the NanoBiT assay; the neighboring B- and D-sites are zoomed in. (D) NanoBiT complementation measured upon co-expression of LgBiT-SURF4 and the indicated SmBiT-SEC24A mutants, normalized to WT. (E) Model summarizing secretion dissected by NanoBiT and pulse-chase. SEC24A engages SURF4 via a D-site-cytosolic loop interaction, whereas the B-site is important for PCSK9 secretion but not for SURF4 engagement. SEC24C engages C-terminal motifs of SURF4 via its B-site to drive Cab45 export. Statistical tests were one-way ANOVA with Dunnett’s correction for multiple testing. Data distribution was assumed to be normal but this was not formally tested. ns = not significant, * = P value <0.033, ** = P value <0.002, *** = P value <0.0002, **** = P value <0.0001. For each NanoBiT experiment, six technical replicates were used in each of the three independent biological replicates, as indicated by differential coloring within superplots. Triangles represent mean and error bars represent SD.