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Sec22b controls VWF trafficking and WPB size
Discussion
SNARE proteins are key drivers of membrane fusion that initiate and regulate specificity of membrane docking and bilayer mixing.30 There is a subcategory of SNARE, known as longin-SNARE (YKT6, VAMP7 and Sec22b), which generally participate in fusion events during bio- genesis, maturation and exocytosis of secretory organelles in eukaryotic cells.43-47 In this study, using a shRNA screen targeting longin-SNARE we identified Sec22b as a novel regulator of WPB formation and VWF trafficking, its depletion leading to short and “stubby” WPB that were accompanied by Golgi disintegration and retention of VWF in the ER.
Sec22b is a known participant in anterograde transport of cargo in the early secretory pathway.48,49 Overexpression of the Sec22b-DSNARE variant led to a dominant-negative effect resulting in smaller WPB, cor- roborating experiments that used RNA interference and CRISPR strategies to deplete Sec22b expression. This is most probably a consequence of Sec22b-DSNARE out- competing endogenous Sec22b while failing to properly bind with its cognate SNARE due to the lack of the SNARE helix,50 which precludes formation of a complete trans-SNARE complex and subsequent membrane fusion. The importance of a functional, fusion-competent Sec22b has also been highlighted by mutations in the SNARE domain of the Drosophila homolog of Sec22b, which caused disruption of ER-Golgi transport and resulted in cargo retention in the ER and abnormal ER morphology.51 Similar ER cargo retention is likely at play in Sec22b- depleted EC in our study (Figures 3 and 4). Congestion of anterograde flux from the ER limits the supply of VWF to the Golgi and as WPB formation is driven by condensation of multimeric VWF in the TGN, we assume this reduction is (at least partly) responsible for the smaller size of WPB.
The smaller WPB phenotype is likely further exacerbat- ed by fragmentation of the TGN (Figure 2), which was previously proposed to limit the possibility of adjacent VWF quanta co-packaging into a single, extended WPB.24 Interestingly, that study also demonstrated that experi- mental reduction of VWF trafficking by small interfering RNA silencing of VWF synthesis led to reduction of WPB length without affecting overall Golgi morphology. The data we present here suggest that a Sec22b-dependent trafficking pathway is used both by VWF and by compo- nents that establish or maintain Golgi ribbon integrity. This is in line with previous studies of Sec22b in other models, such as an Arabidopsis mutant deficient for the Sec22b homolog, which displayed comparable TGN dis- ruption.52 The observed phenotype may be a conse- quence of defective trafficking of direct regulators of Golgi morphology, such as Golgi reassembly stacking proteins (GRASP)53 or golgins.54 Indeed, Golgi fragmenta- tion and the concomitant reduction in the length of WPB has also been observed after depletion of Golgi tethering proteins (GM130, GRASP55 and giantin).24 TGN fragmen- tation may also merely result from the induced imbalance in trafficking, as the phenotype was also reported after disruption of retrograde trafficking from early endosomes or within the Golgi.55,56 While further investigation is required to decipher the precise role of Sec22b in the maintenance of TGN morphology, the resulting conse- quences highlight its indispensable function in ensuring adequate trafficking of VWF and WPB biogenesis.
Upon Sec22b silencing, dilated ER cisternae were observed, accompanied by electron-dense, ribosome- studded rER structures that contained VWF aggregates. There was a striking resemblance with the dilated ER morphology that is observed in response to VWD-caus- ing mutations in VWF that affect the protein’s ability to dimerize and leave the ER.57,58 Sec22b is recruited onto ER-derived COPII vesicles that transfer proteins from the ER to the Golgi, through interactions of its longin domain with Sec23/Sec24 of the COPII-coating complex.59,60 Similar dilated ER phenotypes and ER retention of secre- tory proteins have been described in chondrocytes from sec24d-deficient zebrafish61 and in pancreatic acinar cells from Sec23bgt/gt mice that additionally display lack of zymogen granules.62 This suggests that the rate of COPII- mediated, anterograde ER-Golgi traffic underpins the ability of endothelial cells to shape WPB to their typical elongated morphology.
A recent study identified GBF1 as a dynamic regulator of anterograde VWF trafficking and WPB morphology that, dependent on external/environmental cues, controls the flux of proteins (including VWF) from the ER to Golgi.63 Similar to what we observed after Sec22b silenc- ing, depletion of GBF1 led to accumulation of VWF in the ER and a reduction in the overall state of VWF proteolytic processing. However, a number of important phenotypic differences suggest that GBF1 and Sec22b operate through different mechanisms. Unlike Sec22b, GBF1 depletion did not affect Golgi morphology and, unexpect- edly, resulted in unusually large WPB that remained asso- ciated with or in close vicinity of the Golgi. Despite their reduction in length, WPB in Sec22b-depleted cells normal- ly recruited exocytotic components such as Rab27A and Slp4-a (Online Supplementary Figure S5), contrary to the enlarged WPB in GBF1-ablated cells which failed to acquire post-Golgi cargo and Rab27A and which were secretion incompetent.63 Although their short WPB were still responsive to agonists, Sec22b-depleted EC secreted reduced amounts of VWF through the regulated and basal secretory pathway (Figure 4B and C), which we presume is due to a reduction in WPB pool size. These discrepan- cies emphasize that future studies are needed to clarify how such opposing effects on WPB formation and secre- tion can arise from defects in anterograde ER-Golgi trans- port.
In conclusion, we identified Sec22b as a new regulatory component of the endothelial secretory pathway that con- trols VWF trafficking and the morphology of its carrier organelle the WPB. We propose a model (Figure 5D) in which secretory proteins such as VWF and components that control Golgi morphology utilize a Sec22b-dependent pathway to arrive at the Golgi, where VWF is packaged in elongated WPB with dimensions that are proportional to the size of the Golgi. The reduction in WPB length in the absence of Sec22b is explained by a combination of reten- tion of VWF in the ER and disintegration of the Golgi. Reduced flux of VWF through the secretory pathway ulti- mately decreases the amount of VWF that can be secreted by EC that lack Sec22b function. This highlights the importance of efficient transport of VWF through the secretory pathway prior to its packaging in WPB and iden- tifies Sec22b as a potential determinant of plasma VWF levels. Future studies should address the impact of compo- nents of this protein complex on VWF plasma levels in patients with bleeding and thrombotic disorders.
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