Potential Regulatory Mechanisms of SNARE Disassembly

Even though rapid SNARE-complex disassembly is essential for the cell in order to provide sufficient amounts of free SNAREs to readily replenish SNARE pools after fusion, it is nevertheless of high importance that SNARE disassembly, like all vital physiological processes, is tightly regulated. It should for example be granted that uncontrolled disassembly does not deprive the cell of factors which at the same time are needed for other vital processes. αSNAP as well as NSF have been reported to be involved in various other cellular functions independently of each other, making it feasible that there are mechanisms which inhibit disassembly as soon as one of them becomes limiting. Likewise, since ATP is a fuel needed for a large variety of other

vital cellular processes as well, NSF activity might be restricted under conditions in which ATP levels are low. Furthermore, disassembly of SNARE complexes prior to completion of assembly would, according to current knowledge, prevent final membrane merger and should hence also be regulated by the cell.

SNARE-complex disassembly being mediated by the combined efforts of two proteins brings about the question of whether regulatory mechanisms act on the level of the enzyme, the adaptor or either of them. Finally the SNARE target might also be the targets of regulation. Up to date, several groups found potentially regulating factors, covering all three of these possibilities.

The enzyme – Regulation of NSF activity

For NSF, nitrosylation and phosphorylation, both having an apparent inhibitory effect on its function, have been reported. Phosphorylation at residue Ser-237 by a Serine/Threonine-kinase supposedly results in a hexameric form which does not bind SNAP/SNARE complexes [35]. Likewise, phosphorylation at the tyrosine-residue 83 has been suggested to lead to reduced binding of αSNAP [36]. In vitro phosphorylation has also been witnessed for αSNAP where it apparently led to a ten-fold reduction in SNARE-complex affinity [37].

The adaptor – Regulation on the level of the SNAP

Additionally, proteins able to compete with αSNAP for SNARE complex binding have been described. Amongst these are the so called Complexins (also known as Synaphins) which were originally identified by their interaction with the neuronal SNARE core complex [38, 39].

Complexins The Complexins represent a family of small proteins without folding similarities to any other protein family and are present in all multicellular eukaryotes.

Complexins have originally been reported to displaceαSNAP from SNARE complex when added in excessive amounts, leading to speculations that they might have inhibitory properties with respect to SNARE disassembly [39]. Notwithstanding these findings,in vitro assays directly monitoring SNARE disassembly do not show any negative influence of Complexin 1 or 2 on SNARE-complex disassembly [40].

However, due to a time resolution in the range of several minutes, these experiments are preferably suited for comparing ’all-or-nothing’ disassembly defects rather than minor kinetic differences which could hence not be ruled out. Meanwhile the mode of binding between Complexin and the SNARE core complex has been investigated. An α-helical central stretch consisting of 58 amino acids was found to bind the SNARE complex in an anti-parallel fashion [41], and the structure of complexin bound to the neuronal SNARE complex has been solved [42]. Seemingly contradictory outcomes

of knock-out experiments and over-expression studies in various animals and cell-types have complicated the determination of complexin function, leaving a range of proposed models in the field up to now. Most of these studies report a reduction of exocytosis in presence of excess Complexin which would be in line with a negative impact on fusion. However, at the same time the absence of complexin appears to result in impaired exocytosis as well. Recently, three different studies resulted in findings which led to a similar interpretation, according to which Complexin might act as a so called fusion clamp binding the SNARE complex in a partially zippered state prior to complete fusion and thereby ’clamping’ it until the signal for the final fusion step is received [43, 44, 45]. Even though this scenario primarily envisions Complexin to function as an inhibitor of the last step of SNARE assembly, it does not exclude the possibility that the ’Complexin clamp’ serves as a protection against unwanted disassembly at the same time. Altogether the apparently contradictory results could in principle also be explained by a fine-tuning capability which for instance might depend on a physiological Complexin concentration. If this were to be the case, too little as well as too much Complexin would harm the balanced fusion process and might lead to similar phenotypes. Complexins’ probably complex role and possible implications on SNARE disassembly hence require further elucidation.

The membrane – Another check-point of SNARE regulation?

Even though SNARE complexes readily assemble and with the help of NSF and αSNAP disassemble in solution, they are naturally located on membranes, which might also influence their mechanism as well as speed of assembly or disassem-bly. SNAREs have been inserted into membranes but only little has been done to dissect the process on membranes in detail. What has been done up to now has led to contradictory results. EPR-measurements have attributed a possible role of the membrane to control the capability of SNARE motifs to enter SNARE com-plexes. They indicated that a short membrane proximal region of Synaptobrevin-2 is dipped into the membrane and thereby inhibits the formation of SNARE com-plexes [46], a regulatory mechanism that was called ’Synaptobrevin restriction’ which could in this study be overcome by mutation of two membrane proximal tryptophan residues. This scenario would to some extent contradict the results mentioned above, which suggested that SNARE zippering proceeds in an N- to C-terminal direction.

Notwithstanding these findings, other groups have witnessed fusion of native Synap-tobrevin membranes and SynapSynap-tobrevin has recently been shown to be constitutively active, regardless of whether incorporated into membranes or not [47]. Here Synap-tobrevin could be driven into SNARE complexes both in isolated synaptic vesicles and in proteoliposomes. Even though the membrane thus does not seem to regulate SNARE assembly as such, it might influence one of the other steps of the SNARE

cycle. Recently, Munc-13 was found to interact with SNARE complexes when these are incorporated into liposomes, whereas they hardly interacted in solution [48].

Likewise, SNARE disassembly might be affected by the membrane. αSNAP and its yeast homologue Sec17 have for instance been suggested to directly interact with membranes independently of the well established interaction with membrane bound SNARE complexes, which of course also confines them to the membrane [49, 50].

This is in line with the observation that αSNAP is an amphipathic proteins which binds to plastic surfaces [51]. Steel et al. report that the stimulation of the NSF ATPase-activity byαSNAP is more pronounced in the presence of lipids [50]. Lastly, NSF is released inefficiently from many cellular membranes under conditions which allow for 20S-complex disassembly. This might be an evidence for NSF-binding to lipids as well. Nevertheless, no experiments directly assessing the impact of membranes on SNARE disassembly have been performed so far, leaving room for further investigations.

1.3.6 The Yeast Homologous Protein Family – How