Segregation of the Qb‐SNAREs GS27 and GS28 into Golgi Vesicles Regulates Intra‐Golgi Transport

Abstract

*Corresponding authors: Alexandre A. Mironov, alexandre.mironov@ifom.eu † These authors contributed equally to this work.The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgiresident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb-SNAREs GS27 (membrin) and GS28 (GOS-28), and depleted of nucleotide sugar transporters. A block of intra-Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra-Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP-galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra-Golgi transport; re-addition of isolated Golgi vesicles devoid of UDP-galactose translocase obtained from normal cells restores intraGolgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter-cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of εCOP accelerates the cisto-trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intraGolgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter-cisternal connections.

Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

“…[ 28 ] and Yang et al . [ 32 ] gave identical results; our unpublished observations) were added to the incubation medium, the degree of Golgi tubulation greatly increased ( Figure 5 F, Figure 5 A-magenta bar), and this stimulatory effect was blocked by αSNAPmu ( Figure 5 A-brown bar). Similarly, BFA (2 µg/mL for 90 min) induced tubulation of isolated Golgi membranes, although only in the presence of externally added COPI-dependent vesicles ( Figure 5 G, Figure 5 A-white bar), and not in their absence ( Figure 5 A-black bar).…”

“…Unless otherwise stated, all chemicals and reagents were obtained from the previously indicated sources [ 32 , 38 , 39 , 40 , 41 , 42 ] or from Sigma (Milan, Italy). Anti-βCOP (EAGE), anti-ManII, anti-membrin and anti-GOS28 polyclonal antibodies (pAbs) were purchased from Thermo Fisher Scientific Inc (Rockford, IL, USA), and all were used at a 1:500 dilution.…”

Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

“…At the molecular/mechanistic level, Golgi tubule formation has been proposed to be initiated by COPI coatomer-mediated budding (Yang et al, 2011), and tubule elongation and fission appear to require the actions of cytosolic phospholipase A2 (cPLA2) and lysophosphatidic acid acyltransferase-γ (LPAATγ), respectively (San Pietro et al, 2009) (Yang et al, 2011). Recent evidence also points to a role for Golgi localized SNAREs and BARS in the dynamics of the intercisternal connections (Fusella et al, 2013). Nevertheless, a complete understanding of the molecular players regulating the intra-Golgi connections remains lacking.…”

“…Continuity-mediated transport has been observed to occur between endosomes and lysosomes (Luzio et al, 2007), and also the exocytic release of cargo from secretory granules (Rutter and Hill, 2006) or synaptic vesicles through transient pores (kiss-and-run) (Rizzoli and Jahn, 2007; Alabi and Tsien, 2013) at the plasma membrane can be considered to occur via this modality. For intra-Golgi transport, this mechanism has been discussed several times in the past (Mellman and Simons, 1992; Weidman, 1995; Mironov et al, 1997; Marsh et al, 2004; Trucco et al, 2004; Mironov et al, 2005; Beznoussenko et al, 2007; Glick and Luini, 2011) and a few recent intra-Golgi transport models including the mixing–partitioning (Patterson et al, 2008), the kiss-and-run (Mironov and Beznoussenko, 2012; Fusella et al, 2013; Mironov et al, 2013) and the cisternal progenitor schemes (Pfeffer, 2010) have been proposed that imply transient tubular continuities across cisternae. At the molecular/mechanistic level, Golgi tubule formation has been proposed to be initiated by COPI coatomer-mediated budding (Yang et al, 2011), and tubule elongation and fission appear to require the actions of cytosolic phospholipase A2 (cPLA2) and lysophosphatidic acid acyltransferase-γ (LPAATγ), respectively (San Pietro et al, 2009) (Yang et al, 2011).…”

The Step-Wise C-Truncation and Transport of ESyt3 to Lipid Droplets Reveals a Mother Primordial Cisterna

“…Correlative-light immunoelectron microscopic analysis (CLEM). Fluorescence microscopy was combined with immune EM and three-dimensional correlative electron tomography (Micaroni et al, 2010) (Fusella et al, 2013) (Beznoussenko et al, 2016) to study the ultrastructure of specific segments of type 1 and type 2 primordial cisternae and their association with E-Syt3C2C. The necessary cell fixation with 4% paraformaldehyde and 0.05% glutaraldehyde precluded the use of the anti-E-Syt3 antibodies.…”