Fig 2: ERGIC-53 is a novel interaction partner of BET1 with impaired interaction of Ile51Ser BET1 and ERGIC-53 Volcano plot of AP-MS data using HA-BET1 or Ile51Ser.Co-IPs using HA-tagged constructs expressed in HEK293 cells. As baits HA-tagged proteins were immunoprecipitated with anti-HA beads. IP samples and cell lysates were subjected to immunoblotting with a-HA, a-ERGIC-53, a-BET1, a-SEC22b, a-GOSR2, and a-GAPDH antibodies, respectively.Quantification of three independent replicates of Co-IP experiments. Protein levels of the Co-IP samples were normalized to BET1-HA-levels. Data represented are mean ± SD of three biological replicates. Significant differences were checked by one-way ANOVA followed with Tukey's post hoc test. *P = 0.05.Endogenous Co-IP of BET1 and ERGIC-53. Immunoblots of HEK293T cell lysates (+lysate) or lysis buffer (-lysate) incubated with (+) or without (-) BET1 antibody followed by precipitation with protein A/G beads. ERGIC-53 signal was only present in samples with lysate and antibody, whereas * denotes unspecific signals from beads used for precipitation. As a positive control, GOSR2 antibody was used.Subcellular localization of ERGIC-53 in fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of ERGIC-53 (green) and GM130 (red) in fibroblasts from P1, P2, and a control cell line. Scale bar: 20 µm. PCC (right panel) for GM130 versus ERGIC-53 (C1: n = 74, C2: n = 95, C3: n = 59, P1: n = 72 and P2: n = 76). (***P = 0.001, one-way ANOVA was followed by Tukey’s post hoc test). Shown are means ± SEM. Immunofluorescence images shown are one example of a total of two biological replicates.Colocalization of ERGIC-53 and BET1 in control and P2-derived fibroblasts. Representative immunofluorescence images of ERGIC-53 (green) and BET1 (red) in fibroblasts from P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for BET1 versus ERGIC-53 (C1: n = 72, C2: n = 46, C3: n = 56 and P2: n = 37). (***P = 0.001, one-way ANOVA was followed by Tukey’s post hoc test). Data represented are mean ± SEM of two independent experiments.Immunofluorescence images of control fibroblasts (C1) and patient fibroblasts (P1 and P2) treated with Brefeldin A for 40 min and Golgi recovery for indicated time points. Golgi reconstitution was analyzed by immunofluorescence microscopy of the cis-Golgi marker GM130. Cells displaying an intact Golgi are marked with a plus (+). Scale bar: 20 µm.The number of cells with an intact Golgi was divided by the number of total cells per image and plotted against time (three biological replicates). Dots are means ± SD. For quantification, a sigmoidal fit was performed with the software GraphPad Prism 7. Dashed lines indicate extrapolated plateaus. LogIC50 ± 95% confidence interval: C1 = 59.38 ± 2.31 min, P1 = 72.89 ± 1.65 min, P2 = 68.57 ± 1.929 min. Significant differences were assumed when 95% confidence interval do not overlap. Source data are available online for this figure.

Fig 3: Genetic studies in P1 Pedigree of P1 with corresponding BET1 Sanger chromatogram depicting the paternally inherited c.202G>C; p.(Asp68His) missense variant and a maternally inherited c.134delC; p.(Ala45Valfs*2) frameshift variant (NM_005868), consistent with recessive inheritance.Sashimi plots comparing fibroblast RNA sequencing reads in P1 (red) and two control muscle samples (blue and green) at the exons 3–5 of BET1.Immunofluorescence images of BET1 (red) and GOSR2 (green) in fibroblasts from P1, P2, and control. In P1, BET1 immunoreactivity is reduced. Scale bar: 25 µm.In the overlay, BET1 colocalization with GOSR2 and Syntaxin-5 is reduced in P2 compared to controls while BET1 colocalization with SEC22b is unchanged. BET1 colocalization was determined using Pearson’s correlation coefficient (PCC) on regions of interest (ROIs) drawn around cells (GOSR2 P2: n = 56; C: n = 86, P = 0.0025) (Syntaxin-5 P2: n = 27; C: n = 32 P = 0.0006) (SEC22b P2: n = 27; C: n = 30, P = 0.2093). (*P = 0.05 Mann–Whitney U-test). Data represented are mean ± SEM.

Fig 4: Normal SNARE complex partners in BET1 fibroblasts with reduced colocalization at the cis-Golgi in transfected cells Quantification of protein levels of BET1 SNARE complex partners and the novel interaction partner ERGIC-53 in fibroblasts. Values are normalized to GAPDH. No significant differences between the control and patient fibroblasts were detected. Data represented are mean ± SD of four biological replicates. Significant differences were analyzed by one-way ANOVA followed by Tukey's post hoc test.Immunofluorescence images of BET1 knockdown HeLa pC4 cells transfected with BET1 siRNA #1 10 min after induction of ER-to-Golgi transport with solubilizer. Scale bar: 20 µm.HeLa pC4 cells were transfected with control siRNA (ctrl) or siRNA against BET1 (#1 and #2) and protein levels of BET1 and ER-to-Golgi complex SNAREs SEC22b, Syntaxin-5, and GOSR2 were analyzed by immunoblot. *residual SEC22b signal after immunoblot stripping.Quantification of EV2C. Data represented are mean ± SD of three biological replicates. Significant differences to ctrl cells were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. ***P = 0.001.Table showing a spectral count-based summary of three independent AP-MS experiments with significant hits shown in Fig 5A. Hits are sorted by the difference of BET1-WT and BET1-Ile51Ser. Hits which do not fulfill the criteria to have a mock-transfected spectral count of two or less are colored in grey.