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In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption., (© 2020 Grond et al.)

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D-ceramide-C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D-ceramide-C6-treated HeLa cells revealed an increase in the levels of C6-sphingomyelin, C6-glucosylceramide, and diacylglycerol. D-ceramide-C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D-ceramide-C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6-sphingomyelin prevented liquid-ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.