Fig 2: Pedigrees and clinical presentation of individuals with COPB1 mutations. a Family 1 includes two affected individuals IV2 and IV3. Individuals IV5, IV6 and IV7 are also suspected to have been affected with microcephaly and developmental delay. They died in early childhood and no further details were available. IV2 and IV3 have minor dysmorphism with up-slanting palpebral fissures. b Family 2 includes four affected individuals from two nuclear families all from the same Saudi tribe. c Gel electrophoresis of RT PCR amplicons in Family 1 demonstrates 2 bands in the homozygous state (IV2, an affected individual) and in the heterozygous state (III1, an unaffected parent). d Electropherograms of band A and band B from the heterozygous parent and homozygous proband. In the proband’s trace, a G has effectively been deleted due to the creation of a new donor site by the G>T mutation. In band B, Exon 8 has been skipped (proband and parent)

Fig 3: ERK8 regulated GalNAc-T traffic depends on the activity of COPI regulators.(A) Tn expression levels, as determined by Helix Pomatia Lectin (HPL) staining in cells expressing dominant-negative mutant Arf1 (Q71L) compared to wild-type Arf1. GRASP55 labels the Golgi apparatus. Scale bar: 10 μm. (B) Quantification of Tn expression in ERK8-depleted cells upon treatment with 50 nM GBF1 inhibitor, Golgicide, for the indicated times. (C) Quantification of Tn expression in cells following co-knockdown with ERK8 and GFP or NT5 siRNA in amounts to equivalent to double (GFP coKD), triple (GFPx2 coKD) and quadruple (GFPx3 coKD) siRNA transfection configurations. (D) SDS-PAGE analysis of protein levels of Arfs and GBF1 in single and double knockdown configurations. (E) Beta-COP (COPB) staining in control and ERK8-depleted cells. Scale bar: 10 μm. (F) Quantification of the relative numbers of COPI transport carriers (COPB) and Golgi fragments (GM130) using the granularity measurement module in MetaXpress software. At least 30 cells (non-targeting (NT) control, 37 cells; ERK8-KD, 34) were quantified for each treatment. (G) Staining of native COPI coatomer in cells treated with 5 μM Ro-31-8220 (iERK8) over the indicated times. (H) Quantification of the relative numbers of COPI transport carriers using the granularity measurement module in MetaXpress software. Twenty-five or more cells (Vehicle, 41 cells; 5 min iERK8 treatment, 25; 25 min iERK8 treatment, 57) were quantified for each treatment. (I) Staining of GalNAc-T1 (left panel) and KDEL receptor (KDELR1) localisation (right panel) in HeLa KDEL-R1-GFP stable cell line depleted of ERK8. (J) Quantification of total intensities of GalNAc-T1 and KDEL-R at the Golgi region demarcated by Giantin (Golgi protein) staining. Values on graphs indicate the mean ± SEM. **p<0.0001, *p<0.05 by two-tailed unpaired t test, relative to vehicle treated (B and H), single ERK8 knockdown (C) and NT siRNA-treated (F and J) cells. NS, not significant.DOI: http://dx.doi.org/10.7554/eLife.01828.012

Fig 4: ERK8 regulates COPI-dependent GalNAc-T traffic.(A) Co-knockdown of ERK8 with Arf1 or GTP exchange factor, GBF1, and staining with Helix pomatia lectin (HPL). NT, non-targeting. Scale bar: 30 μm. (B) Quantification of Tn levels upon ERK8 co-knockdown with Arf proteins and GBF1. Grey bar indicates knockdown of ERK8 only. Blue bars indicate co-knockdowns. (C) SDS-PAGE analysis of total Arf and Arf1-GTP in cells treated with 5 µM Ro-31-8220 (iERK8). (D) Co-staining of Beta-COP (COPB) and GalNAc-T1 in cells treated with 5 µM iERK8 for 15 min. Transient tubular structures emanating from the Golgi appear stained for GalNAc-T1 and beta-COP (arrowhead, second panel). Scale bar: 10 μm. (E) Effect of ERK8 depletion on GalNAc-T1 and KDEL receptor (KDEL-R) subcellular location. (F) Effect of expression of active SRC (SrcE-mcherry, containing the E378G mutation) or inactive SRC (SrcK-mcherry, containing the K295M mutation) on both proteins. Scale bar: 10 μm. (G) Visual scoring of KDEL-R and GalNAc-T1 redistribution from the Golgi in cells subjected to various treatment conditions. Cells were counted in each condition from three independent experiments (NT control: 83 cells; ERK8 KD: 86; SrcE-mcherry: 42; SrcK-mcherry: 32). (H) Temperature-sensitive vesicular stomatitis virus G glycoprotein (VSVG-mcherry) traffic to the Golgi induced by shift from restrictive to permissive temperature for 15 min in KDEL-R expressing cells stained for GalNac-T1. Scale bar: 10 μm. (I) Visual scoring of KDEL-R and GalNAc-T1 relocation in VSVG expressing cells at 0 and 15 min after temperature shift. Cells were counted in each condition from three independent experiments (0 min, 44 cells; 15 min, 63 cells). Values on graphs indicate the mean ± SEM. **p<0.0001, *p<0.05 by two-tailed unpaired t test, relative to NT siRNA-treated (B and G) cells and cells at 0-min timepoint (I).DOI: http://dx.doi.org/10.7554/eLife.01828.011

Fig 5: Targeted CRISPR/Cas9 disruption of copb1 exon 8 generates a range of insertion-deletion changes in vivo. Xenopus tropicalis copb1 has 21 exons, including an untranslated first exon (a). CRISPR/Cas9 directed indel formation disrupting X. tropicalis exon 8 using 2 sgRNAs (red: sgRNA3 and sgRNA4 (b)) results in homozygous frameshift and splice changes akin to those identified within the patient subpopulation. Genotyping analysis of X. tropicalis crispants details the range of indels across three groups of 10 pooled tadpoles (NF41) injected with sgRNA3 following PCR amplification and Sanger sequencing of subclones (c). The 41-bp deletion observed in gDNA subclones extends into the intronic region and is proposed to induce exon 8 skipping in crispant X. tropicalis tadpoles. Total RNA was obtained from 4 groups (un-injected control (1), copb1 sgRNA 3 crispant set 1 (2) and copb1 sgRNA 3 crispant set 2 (3), injected control (4)) of 10 pooled individuals as outlined in Guille 1999 [51] and cDNA synthesised using Primer Design’s Reverse Transcription Premix 2. Amplification across the copb1 region of interest revealed a band at 382 bp (exons: 7, 8 and 9) and an additional band in crispants at 262 bp (exons: 7 and 9, Additional file 1: Fig. S1D)