Otros elementos para el diseño y clasificación de

Once a vesicle is tethered to a membrane the flexibility of the tether may permit the vesicle to sample the membrane for the cognate SNARE to facilitate vesicle docking. SNAREs are members of a superfamily of highly conserved proteins (Weimbs et al., 1997, 1998) that reside on vesicles (v-SNAREs) and target membranes (t-SNAREs;

Rothman, 1994; Rothman and Warren, 1994). The central tenet of the SNARE hypothesis, originally formulated with brio and verve by Rothman (Rothman, 1994), is that the v-SNARE on the vesicle binds to its cognate t-SNARE on the target membrane to form a ^m^j'-SNARE pair (trans- denotes that the SNAREs reside in opposite membranes; Bennett et al., 1993a; Sollner et al., 1993b; Protopopov et al., 1993; Pevsner et al., 1994a; Sogaard et al., 1994). This event confers targeting specificity to a transport reaction and is termed vesicle docking. Vesicle docking is distinct from tethering as it involves SNAREs, and brings the membranes into very close apposition. Tethering may be considered to involve links that extend over distances of more than half the diameter of a transport vesicle (>25nm). Whereas, docking brings membranes to within a bilayer’s distance from one another (<5-1 Onm; Pfeffer, 1999). Targeting specificity is ensured by the cognate v-/t-SNARE pair, which can be seen as a ‘lock and key’ device to discrete compartments, and a multitude o f v-SNARE and t-SNAREs have been identified in multiple steps of the secretory pathway from yeast to mammals (Bock and Scheller, 1999; Jahn and Siidhof, 1999; Table 1.3).

The syntaxin family of t-SNAREs and VAMP family of v-SNAREs are type II membrane proteins containing a C-terminal signal anchor, and with most of the mass projecting into the cytoplasm. The SNAREs have been grouped in a superfamily on the basis of having either one or two homologous coiled-coil domains o f c. 60 amino acids, termed the SNARE motif (Weimbs et al., 1997, 1998; Jahn and Siidhof, 1999). This motif is the defining feature of SNAREs and functions in the associations between cognate SNAREs to form the ‘core complex’ that is essential for their function in

vesicle docking and fusion (Fiebig et al., 1999; Parlati et al., 1999). SNARE subfamilies can be defined by whether a SNARE has one or two SNARE motifs, and on the sequences that flank these SNARE motifs. The majority of SNAREs contain one SNARE motif preceded by a variable N-terminal domain which may contain other coiled-coil regions important for inter- and intra-molecular interactions, as in the syntaxins (Hanson et al., 1995). The SNARE motif is then proceeded by a basic domain followed by a C-terminal TMD (Weimbs et al., 1998). This organization is typical of the syntaxin family of t-SNAREs and the VAMP and B etlp/B oslp v-

Chapter 1 Introduction

Table 1.3 Localization and classification of SNAREs.

SNARE Organism Type Localization

P e p l2 p Yeast 0 , t-SNARE Golgi-yacuole/lysosome Vam3p Yeast 0 , t-SNARE Vacuole

Ssolp Yeast 0 , t-SNARE Plasma membrane Sso2p Yeast 0 , t-SNARE Plasma membrane T lg lp Yeast 0 , t-SNARE Late Golgi-endosome Tlg2p Yeast 0 , t-SNARE Late Golgi-endosome Sed5p Yeast 0 , t-SNARE ER-c/5-Golgi

U felp Yeast 0 , t-SNARE ER-cz5-Golgi Sec9p Yeast 2 0 , t-SNARE Plasma membrane G oslp Yeast 0 , y-SNARE ER-Golgi

B oslp Yeast 0 , y-SNARE ER-Golgi B e tlp Yeast 0 , y-SNARE ER-Golgi

V tilp Yeast 0 , y-SNARE ER-Golgi-vacuole Sec22p Yeast R, y-SNARE ER-Golgi

Y kt6p Yeast R, y-SNARE ER-Golgi N y v lp Yeast R, v-SNARE Vacuole

Snclp Yeast R, v-SNARE Plasma membrane Snc2p Yeast R, y-SNARE Plasma membrane

Syntaxin-1 Mammals 0 , t-SNARE Plasma membrane (neurons) Syntaxin-2 Mammals 0 , t-SNARE Plasma membrane

Syntaxin-3 Mammals 0 , t-SNARE Transport vesicles, plasma membrane Syntaxin-4 Mammals 0 , t-SNARE Plasma membrane

Syntaxin-5 Mammals 0 , t-SNARE Golgi, VTCs, ER, COP coated vesicles Syntaxin-6 Mammals 0 , t-SNARE TGN-endosomes

Syntaxin-7 Mammals 0 , t-SNARE Golgi/lysosomes Syntaxin-8 Mammals 0 , t-SNARE ER, endosomes S yntaxin-10 Mammals 0 , t-SNARE TGN

Syntaxin-12 Mammals 0 , t-SNARE Endosomes S yntaxin-16 Mammals 0 , t-SNARE Golgi

rB etl Mammals 0 , v-SNARE Golgi, VTCs, ER, COP coated vesicles GS15 Mammals 0 , v-SNARE Golgi

GOS-28 Mammals 0 , v-SNARE Golgi, COP coated vesicles Membrin Mammals T, y-SNARE ER/Golgi, COP coated vesicles rSec22 Mammals R, y-SNARE ER/Golgi, COP coated vesicles SNAP-25 Mammals 20, t-SNARE Plasma membrane

SNAP-23 Mammals 20, t-SNARE Plasma membrane SNAP-29 Mammals 2 0 , t-SNARE Plasma membrane VAM Pl Mammals R, y-SNARE Synaptic vesicles

VAMP2 Mammals R, v-SNARE Synaptic/clathrin coated vesicles VAMP3 Mammals R, v-SNARE Clathrin coated vesicles

VAMP5/6 Mammals R, y-SNARE Plasma membrane and intravesicular structures (skeletal muscle and heart) Endobrevin Mammals R, y-SNARE Endosomes

A dapted from lahn and Siidhof, 1999.

SNARE families. Alternatively, SNAREs of the SNAP-25/Sec9 family are attached to the membrane by lipid modifications. For example, SNAP-25 contains two SNARE motifs that surround a palmitoylated cysteine rich region (Oyler et al., 1989; Hess et

al., 1992). Similarly, the ER v-SNARE Ykt6p may be inserted into the membrane by farnesylation at a C-terminal CAAX box (Sogaard et al., 1994). Recently, SNAREs have been classified as Q- or R-SNAREs on the basis that a central residue in the SNARE motif is either an arginine or a glutamine (Fasshauer et al., 1998). In the SNARE core complex these residues form an ionic layer in which one R-SNARE binds to three Q-SNAREs (Sutton et al., 1998). Some authors have proposed that the Q-/R- SNARE terminology is preferable to the v-/t-SNARE terminology since the localization of a given SNARE to a vesicle or target membrane does not necessarily correlate with the structurally defined SNARE subfamilies (Fasshauer et al., 1998).

A recent flurry of papers have revealed that, at least in biochemical reactions carried out to equilibrium, SNARE interactions are more promiscuous than would be predicted by the SNARE hypothesis (Rothman, 1994; Fasshauer et al., 1999; Yang et al., 1999; Grote and Novick, 1999; Tsui and Banfield, 2000). This may not be so surprising given the high homology of the SNARE motif between SNAREs, and it may be that trafficking specificity is conferred by the more variable N-terminal domain of SNAREs (Jahn and Siidhof, 1999). However, it is important to note that these non-cognate SNARE interactions have not been demonstrated to be able to support any function. Thus, it is unclear whether they can support vesicle docking, and/or fusion, in any biological context, and so may be irrelevant biochemical anecdotes. It may be that such non-cognate SNARE pairs simply do not form in vivo, due to the myriad of protein- protein interactions that must occur prior to trans-SNARE pairing, and that in fact each level of these protein-protein interactions confers overall targeting specificity to each vesicle transport step (Pfeffer, 1999).

Vesicle docking must be a highly regulated process, were cognate v-/t-SNAREs always able to pair this might lead to all the organelles in the cytoplasm becoming docked, and clustered together, and could even compromise organelle identity (Pfeffer, 1999). In yeast vacuole docking, trans-SNAKE pairing requires vacuole acidification, suggesting a highly regulated process (Ungermann et al., 1999b). The Secl/M uncl8 family of proteins also serve to regulate t-SNARE accessibility, and have been termed t-SNARE

Chapter 1_______________________________________________________ Introduction

protectors (Pfeffer, 1999). These are soluble molecules of c. 65kD that bind to the N- terminus of Q-SNAREs of the syntaxin family. Since a t-SNARE cannot bind a Secl/M uncl8 protein and a v-SNARE simultaneously, it has been proposed that these eomplexes form sequentially (Pevsner et al., 1994a, b; Lupashin and Waters, 1997). t- SNARE protectors may block ^r<2«5-SNARE pairing by sterically hindering access to the SNARE motif. Alternatively, the Secl/M uncl8 molecule may bind to a t-SNARE conformation that interacts only poorly with a v-SNARE. Such a conformation occurs shortly after the action of NSF and a-SNAP (see Section 1.3.5) to dissociate cis-

SNARE pairs {cis- denotes the SNAREs reside in the same membrane; Hanson et al., 1995), and may involve an intramolecular interaction between the N-terminal and C- terminal coiled-coil domains of the syntaxin (Hanson et al., 1995; Kosodo et al., 1998). The Secl/M uncl8 protein may break up this intramolecular interaction, and in so doing serve to signal that this SNARE is ready or primed for another round of transport. In this way the Secl/M uncl8 proteins may act to positively regulate t-SNARE function (Dascher and Balch, 1996), rather than just playing a negative role in preventing v- SNARE association.

The v-SNARE on a vesicle must gain access to its cognate t-SNARE to enable vesicle docking and fusion, and this involves displaeement of the t-SNARE protector from the t-SNARE. Such t-SNARE deprotection may be tightly coupled to the vesicle tethering machinery (Pfeffer, 1999). In particular, the yeast Rabl homologue Y ptlp has been

shown to be required for trans-SNARE pairing between the t-SNARE Sed5p and the v-

SNARE Boslp by transiently interacting with Sed5p and displacing the Secl/M uncl8 molecule Sly Ip from it (Lupashin and Waters, 1997; Rothman and Sollner, 1997). Y ptlp then rapidly dissociates from Sed5p and allows B oslp to bind. This requires Yptlp-GTP, which is then strictly a catalyst in rroMj-SNARE pairing, as it is not consumed by the reaction. Furthermore, the Secl/M uncl8 protein may enhance the reactivity of the cognate t-SNARE for specific Rab-GTP and in so doing regulate trans-

SNARE pairing (Lupashin and Waters, 1997). In addition, since GTP hydrolysis by the Rab is not required for docking or fusion per se GTP hydrolysis may serve as a timer that determines the frequency of membrane docking events (Rybin et al., 1996).

Transport vesicles may then contain proteins that maintain Rabs in the active GTP bound state such that tethering and docking may occur (Pfeffer, 1999). The Rabs then serve primarily to regulate the events of vesicle tethering and docking (Pfeffer, 1999). However, they may also be involved in long range movements of vesicles via the cytoskeleton (Echard et al., 1998; White et al., 1999) and also in ensuring vesicles contain the correct complement of v-SNAREs (Lian et al., 1994). This diverse array of functions may be due to a heterogeneous collection of Rab effectors, whose point of action may be spatially and temporally regulated contingent upon the life cycle stage of the transport vesicle.