Secretory vesicle exocytosis:

1.5.1. SNARE proteins:

Since 1970, it was generally accepted that the final step of regulated secretion occurs via exocytosis. Calcium was a key point for secretory vesicles exocytosis [44].

However, the downstream mechanism was completely unknown for more than 20 years. In 1993, Söllner et al. have shown the importance of 3 new proteins in the fusion of synaptic vesicles with the plasma membrane [45]. These 3 proteins all

belong to the SNARE (Soluble NSF Attachment protein REceptor) family. Two of them are localized in the plasma membrane (SNAP-25 and syntaxin) and the other one in the vesicular membrane (synaptobrevin). According to their localisation there were called v-SNARE for vesicular SNAREs or t-SNARE for targeted membrane SNAREs [46].

SNARE proteins were shown to be cleaved by clostridial neurotoxins, which are inhibitors of synaptic transmission, suggesting a direct link between SNARE proteins and synaptic vesicle exocytosis. Vesicle secretion was also inhibited by clostridial neurotoxins in many other different cell types, such as pancreatic ȕ-cells or acinar cells [47]. To confirm the role of SNARE proteins in vesicle exocytosis, antibodies raised against syntaxin have also been used. In a similar manner than neurotoxins, the use of syntaxin antibodies strongly abolished exocytosis in chromaffin cells [48].

Even if the importance of vesicular SNARE proteins in the exocytosis process was established, the exact role of these proteins in exocytosis was unknown. In 1998, Sutton et al. have shown for the first time by x-ray crystallography, that SNAP-25, syntaxin and synaptobrevin were assembled in a zipper-like manner to form a tertiary complex called core-complex or SNARE-complex [49]. This complex was shown to be very stable, since it cannot be denaturized neither by high temperature nor by the use of SDS. Moreover, the SNARE-complex is resistant to the proteolytic action of botulinic or tetanic neurotoxins, which is not the case for each protein of the SNARE-complex taken separately [50].

The formation of the SNARE-complex is vital for vesicle exocytosis. Indeed, it allows bringing in close proximity vesicles and plasma membrane, which normally repulse each other, due to their negatively charged phosphate groups. This close proximity

induces a destabilisation of the normal conformation of the lipid membrane, allowing the fusion of the secretory granules and the plasma membrane and the subsequent release of the secretory products [51].

Formation of the SNARE-complex is regulated by several proteins. One of them is synaptotagmin, which is strongly supposed to regulate the formation of the SNARE-complex in response to calcium influx. Synaptotagmin was first discovered on the membrane of synaptic vesicles [52, 53]. This protein is composed of an N-terminal transmembrane part and a C-terminal cytosolic part, which contains two calcium-binding domains: C2A and C2B [54]. Calcium-calcium-binding does not seem to induce a conformational modification of the protein but rather a modification of its electric potential. This modification permits to the protein to bind the SNARE-complex via syntaxin and SNAP-25 [55]. Synaptotagmin is also able to bind phospholipids at micromolar calcium concentrations [56]. The capacity of synaptotagmin to bind both SNARE proteins and phospholipids led to the hypothesis that synaptotagmin could be a calcium sensor for vesicle exocytosis [54]. This hypothesis was reinforced by experiments by the drosophila and the mouse, for which synaptotagmin mutants have shown a strong decrease of the synaptic transmission [57]. Moreover, the use of anti-C2A antibodies has also shown an inhibition of calcium-dependant exocytosis in neuroendocrine cells [56].

It has been recently shown that another protein called complexin cooperates with synaptotagmin to control the fusion of secretory vesicles with the plasma membrane.

Indeed, addition of soluble recombinant complexin or overexpression of complexin significantly inhibits membrane fusion [58, 59]. Moreover, it has been shown that

complexin was able to bind the SNARE-complex and avoid the fusion of secretory vesicles [60]. When calcium binds to synaptotagmin, it induces the release of complexin from the SNARE-complex, allowing the fusion of the secretory vesicles with plasma membrane [61].

After vesicle fusion, the SNARE-complex is disassembled to allow the formation of new SNARE-complexes. Two proteins are involved in this process: Į-SNAP and NSF. Thanks to his ATPase activity, NSF is directly able to disassemble the SNARE-complex [62]. NSF does not bind directly to the SNARE-SNARE-complex but requires the presence of adaptor proteins called SNAPs (Soluble NSF Attachment Proteins).

SNAPs can exist in different isoforms in mammals: Į-, ȕ- and Ȗ-SNAP. Į- and Ȗ-SNAP are ubiquitously expressed, whereas ȕ-SNAP is only expressed in the brain [63]. SNAPs are recruited from the cytoplasm to the plasma membrane, where they can interact with the SNARE-complex. SNARE-bound SNAPs can then recruit NSF, which in turn disassemble the SNARE-complex trough the hydrolysis of ATP molecules [64].

1.5.2. Rab proteins:

Another protein family that has also been intensively studied for its role in the secretory pathway is Rab. Indeed, Rab proteins are GTPases that belong to the Ras superfamily. Their role in intracellular trafficking between different subcellular organelles is well known [65]. It also appears that certain Rab isoforms are involved in the secretory pathway [66]. The Rab3 isoform has been specifically studied in neurons, for its role in synaptic vesicle exocytosis [67, 68]. Rab3 is associated with membrane vesicles via modification of its C-terminal part, which consists in the

addition of geranyl-geranyl moieties on two cysteins [69, 70]. Rab3 is able to cycle between an active vesicle-associated GTP-bound form and an inactive cytosolic GDP-bound form. In this active form, Rab3 is involved in the targeting and the docking of vesicles to the plasma membrane [71]. Rab proteins also act on the fusion process itself through protein effectors, such as rabphilin or Noc2. Rabphilin has been shown to interact with the SNARE-complex independently of the presence of Rab3 and to favour vesicle fusion. However, the initial recruitment of rabphilin to the secretory vesicles requires the presence of vesicles associated Rab-GTP [72]. Noc2, another effector of Rab3, has been shown to be involved in the secretion process of endocrine and exocrine pancreatic cells [21, 73]. In addition to Rab3, two other Rab isoforms, Rab27a and Rab27b, have also been shown to be involved in the regulation of the secretion pathway in different cell types, such as neurons, endocrine and exocrine pancreatic cells, PC12 cells or gastric surface mucous cells [74].

Similarly to Rab3, Rab27 effect is mediated by different effectors, such as rabphilin, granuphilin/Slp4-a, exophilin or melanophilin/Slac2-a [74]. As Rab3, Rab27 seems to be involved in the docking step of secretory vesicles to the plasma membrane.

However, several cell types (neurons, pancreatic ȕ-cells, melanocytes) co-expressed Rab3 and Rab27, suggesting a distinct role for this 2 proteins in the secretory pathway [75].