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
