The current paradigm for transport through the secretory pathway is that of bulk flow. In this model, constitutive secretion from the ER through the Golgi apparatus to the cell surface occurs by default i.e. no sorting signal is required. All diversions from this pathway to other compartments must be signal mediated. Initial evidence for a default pathway for secretion was reported by Wiedman (1984), who showed that a bacterial protein, P-lactamase could be secreted from oocytes. Further confirmation for the bulk flow model came from experiments in which a tripeptide of the N-linked glycan motif, Asn-X-Ser/Thr was introduced into cells. This could enter various compartments but on entry to the ER could be modified by the addition of the N-glycan core m otif which confined the molecule to the lumen o f the ER and therefore also the secretory pathway. This molecule was found to be secreted from cells so showing that passage through the secretory pathway to the plasma membrane was not signal mediated, the glycopeptide carrying no other motifs than that for N-linked glycosylation (Wieland, et al., 1987). In addition, recognition signals for diversion from this default route have been found for delivery to lysosomes (von Figura and Hasilik, 1986; Lazzarino and Gabel, 1988), to the regulated secretory pathway (Burgess and Kelly, 1987) and for
recycling of escaped ER proteins (Munro and Pelham, 1987). The bulk flow model does not explain measured differences in the rates of export of endogenous secretory proteins from the ER (Lodish, 1988; Rose and Doms, 1988) though differences are most likely to be accounted for by differential rates of folding.
Early evidence for vesicle mediated transport in the secretory pathway was obtained from pulse chase studies using pancreatic exocrine cells which showed that transport of labelled material between the ER and Golgi apparatus involved small vesicles (Jamieson and Palade, 1967). More recently transport vesicles mediating certain steps of the pathway have been isolated and their specificity demonstrated in vitro (Groesch, et al., 1990; Rexach and Schekman, 1991).
The last few years have seen great advances in our understanding of the budding and fusion events and the identity of many of the molecules involved in these reactions is known. The molecular nature of the transport vesicles and the processes of budding and fusion have been most clearly delineated for those steps in the transport of material between successive compartments of the Golgi stack.
When isolated Golgi stacks are incubated with ATP and cytosol many uniform vesicles form from all cistemae. These 75 nm vesicles are proposed to be transport vesicles since they can be shown to contain the VSV G-protein when Golgi stacks are prepared from VSV infected cells (Orci, et al., 1986). Transport of this G-protein in the stack is monitored by changes in glycosylation.
The transport vesicles are either coated with an electron dense material (not clathrin) or uncoated. The use of various inhibitory agents such as
permitted stages of the process to be distinguished (Click and Rothman, 1987; Melancon, et al., 1987). The present model suggests that coat proteins assemble on a Golgi cisterna where coated vesicles form. The vesicles bud, lose their coat and can then fuse with the acceptor compartment (Fig. 1.4). The Golgi-derived coated vesicles have been purified and components of the coat analyzed. The major coat proteins C œ P s') are a-CO P (170 kDa), p-Œ )P (110 kDa), ^ C O P (99 kDa), Ô- COP (61 kDa) and a small GTP binding protein called ADP ribosylation factor (ARP) of 21 kDa (Malholtra, et al., 1988; Serafini, et al., 1991a; Serafini, et al., 1991b). It is suggested that several of these COPs are recruited to the membrane, from cytosolic pools, by the ARP -GTP complex (Orci, et al., 1991; Serafini, et al., 1991a).
The signal for a bud to form at a particular site is as yet not known. The reaction requires ATP but whether the formation of a vesicle is driven by conformational changes in coat proteins or whether the material to be transported reaches a threshold concentration in the region which then triggers bud formation is not yet determined (Orci, et al., 1986).
The rab family of small GTP-binding proteins (which include the yeast, ypt proteins) appear to be localized to distinct compartments. Their differential localization has given rise to the idea that rab proteins are involved in determining the specificity o f a compartment and thus somehow involved in the targeting of vesicles to the correct location (Bacon, et al., 1989; Zahraoui, et al., 1989; Plutner, et al., 1991). More recently, the trimeric G proteins have also been shown to affect secretion and they may be involved in regulating the rate of bulk flow (Barr, et al., 1991; Balch, 1992). The G proteins are known to be involved in intracellular signalling from the cell surface and may
Figure 1.4. The steps involved in a round of vesicular transport
A) The figure depicts a simplified version of the steps involved in a round o f vesicular transport. Successive steps of (1) assembly of coated buds, (2) pinching off, (3) targetting, (4) uncoating, and (5) fusion constitute a round of transport
Known requirements and inhibitors are indicated. AIF
4
" = aluminium fluoride, NSF = NEM sensitive fusion protein, SNAP = soluble NSF attachment proteins.# = soluble protein being transported; # = coat proteins
B) Several of the molecules thought to be involved central to the fusion reaction have recently been isolated. The figure shoes how these molecules might be brought together to ensure specificity of the targeting reaction. Each fusion step would require a different v-snare and t-snare pairing.
A) 1 ATP cytosol acyl-COA
C
3
NSF ATP SNAP acyl-CoA NEM B) t-SNARE v-SNARE 34integrate the rate of secretion with the rate of protein synthesis and the need for cell growth.
Recent findings have greatly increased our understanding of the mechanism of vesicle fusion. Several of the molecules which allow this reaction to occur with the necessary specificity have now been reported. The N-ethylmaleimide-sensitive fusion protein (NSF) was purified on the basis of its ability to restore intercisternal Golgi transport in a cell free assay (Fries and Rothman, 1980; Balch, et al., 1984). It is required for membrane fusion since vesicles accum ulate at the acceptor membrane in its absence (Malholtra, et al., 1988; Orci, et al., 1989). NSF requires additional cytoplasmic factors to attach to Golgi membranes. Three species of soluble NSF attachment proteins (SNAPs) have been purified, termed a , p, y SNAP (Clary and Rothman, 1990a).
SNAPs bind to distinct sites in membranes which have recently been purified from bovine brain and called SN A REs (for SNAP receptors)(Sollner, et al., 1993). These SNAREs when cloned and sequenced were shown to be the previously isolated proteins, syntaxin and synaptobrevin (Sudhof and Jahn, 1991; Bennett, et al., 1992). This has led to the proposal o f a model for vesicle targeting and fusion (Fig. 1.4B). The vesicle is thought to carry a receptor, v-SNARE (synaptobrevin-like) and the acceptor com partm ent a t-SNARE (syntaxin-like). These can specifically pair in a reaction mediated by NSF and SNAPs. Only when appropriate binding is achieved can the fusion of the membranes occur. Each stage of transport is proposed to have a different pair of v- and t-SNAREs so ensuring specificity of fusion at each step.