Vesicular trafficking – Common mechanisms between all transport vesicles

As mentioned above, different proteins participate in vesicle formation, depending on the localization of the vesicle. However, the molecular mechanisms governing the initiation of vesicle formation, the vesicle budding, the detachment of vesicle from the donor compartment, the targeting and the fusion of the vesicle to its target compartment, are common between all the transport vesicles and are described in the following paragraphs.

a) Vesicle formation

Vesicle formation starts with the self-assembly of coat proteins (COP), recruited from the cytosol to the membrane of the donor compartment. The polymerization of coat proteins drives the deformation of the membrane, from a flat patch to a spherical bud. Small guanine triphosphatases (GTPases) regulate the polymerization or the depolymerization of coat proteins from membranes, according to their activation state.

Small GTPases are considered as binary switches, as the GTP-bound form drives the assembly of the coat proteins (active form), while the GDP-bound form induces the disassembly of the coat (inactive form). GTPases cycle between an active and inactive state thanks to the regulators GEFs (guanine exchange factors) and GAPs (GTPase activating proteins) (Nie, Hirsch, & Randazzo, 2003). GEFs catalyse the exchange of GDP with GTP, inducing a conformational change, making small GTPases active. GAPs catalyse the hydrolysis of the GTP into GDP, turning off the small GTPases activity. The small GTPase cycle is presented Figure 8, with the example of Sar1, the small GTPase triggering the formation of COPII-coated vesicles, as explained below.

Figure 8: small GTPase cycle, the example of Sar 1. The Guanine Exchange Factor (GEF) exchanges GDP associated with Sar1 with cytosolic GTP. This turns the inactive Sar1 into its active state. On the contrary,

Once activated, small GTPases recruit COP components to the flat, pre-budding membrane, inducing a deformation of the membrane that ultimately gives birth to the vesicle.

A lot of efforts have been made to identify proteins involved in the scission of vesicles, i.e. the release of the vesicle from the donor compartment. However, no specific effectors have been identified so far. Indeed, vesicles were described to be able to bud in vitro simply in the presence of purified coat components (Matsuoka et al., 1998). It is therefore thought that the vesicle fission is driven by the polymerization of the coat itself (Juan S. Bonifacino & Glick, 2004; Kirchhausen, 2000). A more recent study showed that the vesicle fission was not dependent on GTP hydrolysis from the small GTPases (Adolf et al., 2013).

After budding of the vesicle, the coat of the newly formed vesicle is removed, most probably to facilitate the fusion with the acceptor compartment. The hydrolysis of GTP achieved by the small GTPases leads to the disassembly of the coat just after the scission, allowing the retrieval of the coat components for another round of budding.

Recent studies suggest that the depolymerisation of the coat components (complete or not) may occur later than initially believed, at least until the initiation of the tethering with the target compartment (Trahey & Hay, 2010).

b) Vesicle targeting and fusion

Vesicle targeting and fusion is a highly regulated process. It involves notably tethering factors called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Most of the 38 SNARE proteins found in the human genome are transmembrane proteins, containing a linker domain, one or two SNARE motifs (approximately 70 aminoacids in size) and are present at the surface of either vesicles or their target compartment. The very first SNAREs identified were Syntaxin1, SNAP25 and VAMP proteins, and were described in neurons in the late nineties. Most of the SNAREs are specific to cellular compartments.

Originally, they were classified as v-SNAREs (located at the surface of vesicles) and t-SNAREs (located at the surface of the targeted compartment). The interaction of a v-SNARE with a t-SNARE induces the so-called trans-SNARE complex, where four SNARE motifs form a coiled-coil motif, bringing two membranes closer and eventually contributing to their fusion. SNAREs are now classified based on their crystal structure (Fasshauer, Sutton, Brunger, & Jahn, 1998) and the observation that SNARE motifs contain either an arginine residue (R) or a glutamine residue (Q) at a critical position.

Therefore, the four SNARE motifs leading to a functional SNARE complex are composed by one SNARE motif and three Q-SNARE motifs. With a few exceptions, most of the R-SNAREs are v-R-SNAREs and most of the Q-R-SNAREs are t-R-SNAREs.

The SNARE motifs drive the formation of the SNARE complex. Although a SNARE motif is unstructured in its monomeric form, it becomes highly stable when associated with other SNAREs motifs. In the current model, the assembly of SNAREs is in a ‘zipper’

manner, from the N-terminal to the C-terminal end, which brings the membrane from the vesicle and from the targeted compartment in close contact, initiating therefore their

fusion (Jahn & Scheller, 2006; Lin & Scheller, 1997; Otto, Hanson, & Jahn, 1997). After a series of intermediate steps where membranes merge in a stepwise manner (Figure 9), an aqueous pore is formed, which connects the lumen of the vesicle and of the targeted compartment. Briefly, the assembly of the SNARE complex exerts a mechanical force on the membranes, transmitted by the rigidity of the linker domain. This induces the bending and the deformation of the membrane in close contact, facilitating the formation of fusion stalks (Risselada & Grubmüller, 2012).

After fusion, assembled SNAREs form a stable complex in the resulting fused membrane, and must be mechanically separated. The disassembly is performed by NSF (N-ethylmaleimide-sensitive factor), together with the α-SNAP (soluble NFS attachment proteins) cofactor, which both bind the SNARE complex (Ryu et al., 2015; Söllner, Bennett, Whiteheart, Scheller, & Rothman, 1993; Zhao et al., 2015). When bound to ATP, NSF exhibits a split washer shape (Zhao et al., 2015) that becomes flat when bound to

Figure 9: SNAREs-mediated vesicle fusion. (1) R-SNAREs are on the vesicle whereas Q-SNAREs are on the target compartment. (2) SNARE proteins interact with each other, in a ‘zipper’ manner, forming a trans-SNARE complex. (3) Membranes of the vesicle and the target compartment are close enough to

ADP. This movement induces a conformational change of α-SNAP, forcing a shear motion, which disassembles the SNARE complex. All the proteins involved are then ready for a new cycle of fusion.