GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation

Abstract

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.

Fig. 1

GORAB interacts directly with the NTK domain of Scyl1. a Yeast two-hybrid assay between GORAB and Scyl1 constructs. Prey (AD-GORAB or AD-Scyl1) and bait (BD-Scyl1 or BD-GORAB) constructs were co-transformed into yeast and grown on double-drop-out (DDO) medium to verify expression of both proteins in transformants and on selective quadruple drop-out (QDO) medium to examine protein–protein interactions. Constructs containing AD and BD only were used as negative controls. b Pull-down assays with recombinant GORAB. Top panel: pull-down using bacterially expressed GST and GST-GORAB as bait and sHeLa cell lysate. Bottom panel: pull-down using GST, GST-tagged GORAB or Syntaxin 1 as bait and rat liver Golgi (RLG) membrane extract. Samples were blotted with the indicated antibodies. I input (5%), U unbound fraction (5%), B bound fraction (50%). c Pull-down assay using purified GST-Syntaxin 1 or GST-GORAB as bait and MBP-tagged Scyl1. Samples were subjected to SDS-PAGE and analyzed by Coomassie Blue staining. GST-tagged proteins are marked with an asterisk. BSA used as a carrier protein is marked with a circle. The faint bands running under MBP-Scyl1 correspond to likely degradation products or bacterial contaminants. d Mapping the binding site for Scyl1 on GORAB. Left, pull-down assay using purified GST-tagged GORAB fragments as bait and MBP-tagged Scyl1. Samples were subjected to SDS-PAGE and Coomassie Blue staining. GST-tagged proteins are marked with an asterisk. Right, schematic diagram of GST-tagged GORAB truncation constructs. e Mapping the interaction site for GORAB on Scyl1. Left, cell lysates obtained from cells transiently expressing GFP-tagged Scyl1 constructs were used for a pull-down assay with GST or GST-GORAB as bait and analyzed by western blotting. GST-tagged bait proteins and GFP-tagged proteins in inputs are marked with black or red asterisks respectively. Right, a schematic diagram of GFP-tagged Scyl1 truncation constructs. CC coiled-coil region, COPI COPI binding motif

Fig. 2

GORAB co-localizes with Scyl1 and COPI in discrete domains at the trans-Golgi. a Analysis of GORAB localization at the Golgi. Human dermal fibroblasts were fixed and labeled with antibodies to GORAB, GM130 and TGN46. Scale bar, 10 µm. The linescan is representative of data from n = 20 cells. b Analysis of GFP-GORAB localization in stably transfected HeLaM cells (top) and in HeLaM cells transfected with GORAB siRNA (bottom). Cells were fixed and labeled with antibodies to GORAB (bottom row only) and TGN46. Scale bar, 10 µm. c GORAB Golgi localization using STED microscopy. Human dermal fibroblasts were fixed and labeled with antibodies against GORAB and TGN46. Scale bar, 1 µm. d, e Representative EM micrographs depict localization of GORAB in HeLa cells (d) and human dermal fibroblasts (e). G Golgi. Scale bars, 500 nm. f Co-localization analysis of GORAB, Scyl1 and β’-COP using STED microscopy. Human dermal fibroblasts were fixed and labeled with antibodies against Scyl1, GORAB and β’-COP. Scale bar, 200 nm. Yellow arrowheads mark GORAB puncta co-localizing both with Scyl1 and β’-COP, magenta arrowheads mark GORAB puncta co-localizing with Scyl1 only and cyan arrowheads mark Scyl1 puncta co-localizing with ⍰’-COP but devoid of GORAB. g Co-localization analysis of GORAB and Rab6 using STED microscopy. Human dermal fibroblasts were fixed and labeled with antibodies against GORAB and Rab6. Top, scale bar, 5 µm, bottom, scale bar, 200 nm

Fig. 3

GORAB domains are stable entities. a Fluorescence recovery after photobleaching. Left, FRAP recovery curves for GFP-GalNAc-T2, GFP-GORAB and GFP-Scy1. Means with SEM for GFP-GalNAc-T2 (n = 27 cells), GFP-GORAB (n = 28 cells) and GFP-Scyl1 (n = 18 cells). Dotted lines mark points of half-time recoveries. Right, representative HeLa GFP-GalNAc-T2, HeLaM GFP-GORAB and HeLaM GFP-Scyl1 cells at pre-bleached and selected post-bleached states. Bleached region of interests are marked with yellow boxes. Scale bar, 10 µm. b Localization of GORAB in Scyl1-depleted cells. HeLa cells transfected with control or Scyl1 siRNA were fixed and labeled with antibodies to Scyl1 and GORAB. Scale bars, 10 µm and 1 µm. GORAB domains are marked with yellow arrowheads. c Localization of GORAB in BFA-treated cells. HeLa cells were exposed to 5 µg/mL BFA for 7 min prior to fixation and labeling with antibodies to Scyl1 and GORAB. Scale bars, 10 µm and 1 µm. GORAB domains are marked with yellow arrowheads

Fig. 4

Effect of pathogenic missense mutations upon GORAB behavior. a Location of known missense and single base deletion GORAB mutations in GO patients. Coiled-coil domains are depicted as orange rectangles. b Interaction of GORAB variants with GST-tagged bait proteins, as indicated, using cell lysates from RPE-1 cells expressing the indicated GFP-tagged GORAB variants. Inputs (5%) and bound fractions (50%) were blotted for GFP. c Surface plasmon resonance analysis of GORAB-Scyl1 binding. Top, experimental setup with a GLH sensor chip, cross-linked anti-MBP antibody, MBP-Scyl1 as bound ligand and GST-GORAB variants as analyte. Bottom, binding of GST-GORAB variants at 30 nM concentration for 120 s followed by 600 s disassociation. Similar results were obtained in three separate experiments. d Golgi localization of GFP-tagged GORAB variants using STED microscopy. RPE-1 cells were fixed and labeled with TGN46 antibodies. Scale bar, 5 µm. e COPI subcellular localization in HeLaM cells transfected with GFP or GFP-GORAB and incubated for 10 min with 5 µg/mL BFA. Cells were labeled with antibodies to β’-COP and GM130. Scale bar, 10 µm. Dotted line marks the nucleus. f Quantification of COPI retention in the Golgi region from e. Error bars represent mean ± SD, n = 100 cells in each of 3 independent experiments, *p ≤ 0.05 and ***p < 0.001, unpaired t-test. g COPI subcellular localization in HeLaM cells transfected with GFP or GFP-Scyl1 fixed 10 min after incubation with 5 μg/mL BFA. Cells were labeled with antibodies to β’-COP and GM130. Scale bar, 10 µm. Dotted line marks the nucleus. h Quantification of COPI retention in the Golgi region from g. Error bars represent mean ± SD, n = 100 cells in each of 3 independent experiments, ***p < 0.001, unpaired t-test. i Co-localization between GFP-Scyl1, β’-COP and GORAB in HeLaM cells fixed 10 min after incubation with 5 μg/mL BFA. Cells were labeled with antibodies to GORAB and β’-COP. Scale bar, 10 µm. Dotted line marks the cell boundary. The white line indicates the pixels used for the RGB fluorescence intensity profile plot on the right, which is representative of data from n = 20 cells

Fig. 5

GORAB, via Scyl1, is sufficient to recruit COPI to membranes. a A schematic diagram depicting the mitochondrial relocation assay where the addition of rapamycin induces mitochondrial relocation of FKBP-tagged GORAB, allowing for recruitment of associated factors to this compartment. b Relocation of GORAB-mycFKBP and co-expressed GFP-Scyl1 to mitochondria. HeLaM cells co-transfected with mito-FRB and GORABK190del-mycFKBP constructs were pretreated with 2.5 µg/mL nocodazole for 2 h and further incubated with 1 µM rapamycin or DMSO for 3 h prior to fixation. Cells were labeled with antibodies to myc and mtHsp70. c Relocation of endogenous Scyl1 to mitochondria by GORAB-mycFKBP. HeLaM cells co-transfected with mito-FRB and GORAB-mycFKBP and treated as described in b and labeled with antibodies to endogenous Scyl1 and the Golgi marker β4GalT1. d Relocation of COPI to mitochondria by GORAB-mycFKBP. HeLaM cells co-transfected with mito-FRB, GORABK190del-mycFKBP and GFP-Scyl1 were treated as in b, and additionally with 5 µg/mL BFA for 15 min (lower panel only). Cells were labeled with antibodies to β’-COP and myc. In b–d, scale bars are 10 µm and white lines indicate the pixels used for the RGB fluorescence intensity profile plots shown on the right, which are representative of data from n = 20 cells

Fig. 6

Scyl1-dependent recruitment of COPI to artificial membranes. a Liposome recruitment assay with purified MBP-Scyl1, myr-Arf1 and recombinant coatomer. Inputs (2%) and membrane-bound fractions (40%) were subjected to SDS-PAGE and blotted for MBP, ε-COP and Arf1. b Liposome recruitment assay with purified MBP-IPIP27A (as negative control), MBP-Scyl1 and myr-Arf1 and recombinant coatomer. Inputs (2%) and membrane-bound fractions (40%) were subjected to SDS-PAGE and blotted for MBP, δ-COP, ε-COP and Arf1. c Quantification of recruitment of Arf1 and coatomer (δ-COP, ε-COP) to liposomes in the presence of MBP-IPIP27A and MBP-Scyl1. Error bars represent mean ± SD, n = 6 independent experiments, **p < 0.01, ***p < 0.001, unpaired t-test

Fig. 7

Loss of GORAB causes defective terminal N-glycosylation of proteins. a N-glycome analysis of WT and GO fibroblasts. Quantification of relative intensities of MALDI-TOF-MS signals for N-glycan species detected in lysates from wild-type (N = 3 cell lines) and GO fibroblasts (N = 4 cell lines). Error bars represent the mean ± SEM from 4 independent experiments, *p < 0.05, unpaired t-test. GlcNAc N-acetylglucosamine, NeuAc N-acetylneuraminic acid, NeuGc N-glycolylneuraminic acid. Yellow shading indicates differences between WT and GO fibroblasts. b Analysis of sialylated plasma membrane proteins in WT and GO fibroblasts using MAL and SNA lectins. Top, glycan chains recognized by the lectins. Bottom, non-permeabilized fibroblasts stained with FITC-conjugated lectins. Scale bar, 10 µm. c Quantification of fluorescence intensities from b (150 cells analyzed per cell line in each of 3 independent experiments, min to max box and whisker plot, **p < 0.01, Mann–Whitney U test. d Representative flow cytometry histogram of WT and GO fibroblasts (N = 3 cell lines) stained with FITC-conjugated MAL and SNA lectins. e Analysis of metabolic labeling of WT and GO fibroblasts with alkynyl-tagged sialic acid precursor ManNAl. Co-cultured WT and GO cells were incubated with ManNAI for 10 h, fixed and labeled with antibodies to GORAB and TGN46. Scale bar, 10 µm. f Quantification of ManNAI labeling assessed as fluorescence intensity against that of a Golgi marker, with 300 cells analyzed per cell line in 3 independent experiments, min to max box and whisker plot, ***p < 0.001, Mann–Whitney U test. g N-glycome analysis of control and Gorab^Null mouse skin tissue. Symbols representing monosaccharide residues are as in a. Yellow shading indicates N-glycans different between control and Gorab^Null samples. h, i Left, lectin blot analysis of skin lysates of control and Gorab^Null E18.5 embryos with E-PHA (h) or SNA lectin (i). Right, quantification of E-PHA (h) or SNA (i) levels. Error bars represent the mean + SD, n = 4 independent experiments, *p < 0.05, **p < 0.01, unpaired t-test. In a, g, the glycan colored symbols are drawn according to the Symbol Nomenclature For Glycans convention. The structures shown are those most probable for compositions determined from accurate m/z measurements on the basis of the well-accepted biosynthetic route for N-glycans. Glycan assignments and accompanying masses in g are shown in Supplementary Table 1

Fig. 8

Loss of GORAB alters SialylT localization and Golgi ultrastructure. a Knock-down of GORAB, Scyl1 and Cog3 proteins in HeLa SialylT-HRP cells. SiRNA-transfected HeLa SialylT-HRP cells were lysed, subjected to SDS-PAGE and blotted for GORAB, Scyl1, Cog3 and GAPDH. b Representative EM micrographs depict localization of SialylT-HRP, detected using the DAB reaction to generate electron dense product, in HeLa SialylT-HRP cells transfected with the indicated siRNAs. Scale bar, 500 nm. c Quantification of SialylT-HRP distribution in siRNA-treated HeLa SialylT-HRP cells (n = 22 cells per condition, ***p < 0.001, chi-square test). d Representative conventional thin section EM micrographs of Golgi ultrastructure in WT (n = 4 cell lines) and GO fibroblasts (n = 3 cell lines). Enlarged profiles within Golgi cisternae are marked with a red asterisk. Scale bar, 500 nm. e Quantification of cells with dilated cisternae. Error bars represent mean ± SD, n = 25 cells per cell line, **p < 0.01, chi-square test. f Proposed model for GORAB function in COPI-mediated trafficking at the trans-Golgi. (I) GORAB oligomers are stably associated with the trans-Golgi membrane, forming discrete domains, while GTP loading of Arf GTPase leads to its association with the membrane. (II) Membrane-associated GORAB oligomers recruit Scyl1 and locally concentrate GTP-bound Arf in the domains, facilitating the efficient recruitment of coatomer by coincident detection. (III) Coatomer accumulates in the domains and begins to self-assemble. (IV) Coatomer assembly leads to cargo incorporation into a newly forming COPI vesicle. (V) GORAB may stabilize coatomer assembly by remaining associated with the bud neck during vesicle formation. (V) The completed COPI vesicle detaches from the membrane alongside Scyl1, while GORAB stays at the membrane ready to initiate the biogenesis of a new COPI vesicle