Invited Symposium: Intracellular Traffic of Organelles



Materials & Methods


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Characterization of Drosophila SNAREs, alpha-SNAP and NSFs

Contact Person: Mahmood Mohtashami (moody@zoo.utoronto.ca)


Eukaryotic cells have an elaborate transport system through which proteins and cargo are delivered to the appropriate cellular compartment, or alternatively are secreted (1). It is thought that most if not all transport events are mediated through vesicles budding off from a donor organelle and then fusing specifically to a target organelle. The mechanism controlling membrane fusion has been the subject of intense investigation which has led to the discovery of a set of proteins, conserved from yeast to mammals, that appear to be involved this process (2). Major components of the fusion machinery include the ATPase, N-ethylmaleimide sensitive factor (NSF) and its adaptors, the soluble NSF attachment proteins (SNAP) (3, 4). Both NSF and SNAP are soluble proteins that interact with a family of membrane proteins, collectively termed SNAP receptor (SNARE) proteins (5). This interaction is the basis for the mechanism of fusion as explained by the SNARE hypothesis (6): primed by the catalytic action of NSF and SNAP (7), SNARE proteins present on the vesicles (v-SNARE) form a high affinity complex with their counterpart proteins on the target membrane (t-SNARE), which will lead to the fusion of the juxtaposed membranes (8). One of the predictions of the SNARE hypothesis is that correct targeting of a vesicle to its appropriate target membrane is mediated by the proper pairing of v- and t- SNARE proteins (9). This would require the presence of different SNARE isoforms, particular to each organelle along the transport pathway (10, 11). While many SNARE proteins have been identified through searches of cDNA sequence databases, the role of most of these proteins in transport remains to be proven (12).

The role of SNARE proteins in membrane fusion has been most clearly defined in the compartment at which they were originally discovered: the presynaptic nerve terminal. The synaptic v-SNARE, vesicle associated membrane protein (VAMP, also known as synaptobrevin) (13) forms a highly stable complex with its t-SNARE counterparts, syntaxin (14) and SNAP-25 (15), known as the "7S complex" because of its sedimentation rate in glycerol gradients (16). In this complexed state, the synaptic SNARE proteins are resistant to the action of the clostridial toxins (17, 18, 19, 20) which otherwise, are individually able to proteolyse their target synaptic SNARE protein in the monomeric form and severely inhibit neurotransmission (21, 22, 23). The 7S complex remains associated even in the presence of 1% sodium dodecyl sulfate (SDS) and runs on denaturing polyacrylamide gel electrophoresis, as the sum of the molecular weights of VAMP, SNAP-25 and syntaxin (19). The 7S complex is thought to represent a post-fusion state when the v- and t-SNAREs are all present on the same membrane (20). It is not known exactly when SNAP and NSF are recruited to the membrane, but biochemical studies have shown that NSF is able to dissociate the complex by modulating the structure of the SNAREs, utilizing ATP hydrolysis as its source of energy (24, 25, 26). The disassembly may enable each SNARE protein to be properly sorted or be activated to take part in subsequent fusion events.

A strong case for the SNARE hypothesis may be made in the yeast Saccharomyces cerevisiae where mutations have uncovered several genes in the secretory pathway, including NSF, SNAP and many SNAREs (27, 28, 29). For highly purified vacuolar vesicles, for example, involvement of NSF and SNAP was directly demonstrated when fusion could only occur by pre-incubation of vesicles with yeast cytosol containing wild type but not mutated sec17p or sec18p (yeast SNAP and NSF respectively) (30). Later, it was shown that specific cognate SNARE proteins are required to be present on the vesicles for vacuolar fusion to occur (31). Meanwhile biochemical evidence has accumulated indicating that similar to synaptic SNAREs, SNARE proteins in the ER to Golgi pathway in the yeast directly bind one another (32). The universality of the SNARE hypothesis is further underlined by functional substitution of Sec 17p for mammalian SNAP in an intra-Golgi transport assay (28). This suggests a direct relationship between the yeast’s constitutively operating mechanism of transport and the highly regulated secretion in the mammalian system and higher animals in general.

We have elected to study the process of neurotransmission in Drosophila melanogaster because similar to yeast, many powerful genetic tools are available in this model system to facilitate our investigation yet it possesses the tight regulation of mammalian secretion. Already Drosophila homologs of VAMP (33), SNAP-25 (34) and syntaxin (35) have been cloned. Botulinum toxin has the same inhibitory effect on fly neurotransmission as it does on the mammalian system, suggesting that homologous sets of proteins are involved in neurotransmission (36). This is further confirmed by the discovery of a temperature-sensitive syntaxin mutation in flies that results in immediate paralysis upon shift to non-permissive temperatures (37). Another temperature-sensitive paralytic mutation called comatose has been mapped to the Drosophila NSF-1 (dNSF-1) gene (38). comatose, however, has a much slower rate of paralysis and recovery, suggesting that its function is not immediately required for fusion (37). Interestingly, we have also discovered a second NSF homolog, dNSF-2, a plurality unique to Drosophila (39). The presence of two NSF homologs raises the possibility that individual isoforms may have functions specialized for specific transport steps or cell types. Here, we describe studies aimed at examining the roles of NSF, SNAP and SNARE proteins in Drosophila, drawing parallels between phenotypes of mutant flies and the biochemical interaction of proteins.

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Mohtashami, M.; Peng, X.R.; Trimble, W.S.; (1998). Characterization of Drosophila SNAREs, alpha-SNAP and NSFs. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/klip/mohtashami0771/index.html
© 1998 Author(s) Hold Copyright