BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy (2021)
Figure 5

Figure 5. ERGIC-53 is a novel interaction partner of BET1 with impaired interaction of Ile51Ser BET1 and ERGIC-53.

(A) Volcano plot of AP-MS data using HA-BET1 or Ile51Ser.

(B) 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 α-HA, α-ERGIC-53, α-BET1, α-SEC22b, α-GOSR2 and α-GAPDH antibodies, respectively.

(C) 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.

(D) Endogenous Co-IP of BET1 and ERGIC-53. Immunoblots of HEK 293T cell lysates (+ lysate) or lysis buffer (-lysate) incubated with (+) or without (-) BET-1 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.

(E) 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.

(F) 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.

(G) 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.

(H) The number of cells with 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.

Table 1. Patient's clinical presentation and BET1 variants identified

c.202G>C; p.Asp68His

Paternal

(hg19) chr7:93623697C>G

(hg38) chr7:93994385C>G

c.134delC

p.Ala45ValfsTer2

Maternal

(hg19) chr7:93628492delG

(hg38) chr7:93999180delG

c.152T>G; p.Ile51Ser

Homozygous

(hg19)

chr7:93625626A>C

(hg38)

chr7:93996314A>C

Respiratory failure

(4 months)

Respiratory insufficiency

(4 months)

Delayed speech with poor articulation, progressive and refractory epilepsy

(4 years)

Mild tracheomalacia and mild bronchomalacia

1Transcript ID: NM_005868.6; 2 Genome aggregate database allele frequency;3 Combined Annotation Dependent Depletion. NP = not performed

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