Invited Symposium: Intracellular Traffic of Organelles



Materials & Methods


Discussion & Conclusion



INABIS '98 Home Page Your Session Symposia & Poster Sessions Plenary Sessions Exhibitors' Foyer Personal Itinerary New Search

Characterization of Drosophila SNAREs, alpha-SNAP and NSFs

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

Discussion and Conclusion

Syntaxin, VAMP and SNAP-25 assemble into trimeric, SDS-resistant complexes, forming receptors for SNAP, the adaptor protein for the ATPase, NSF (5). It has been shown that NSF is able to dissociate these complexes through ATP hydrolysis. In order to test this function in Drosophila NSFs, we first demonstrated the formation of mammalian SDS resistant complex using recombinant proteins (fig. 1), expecting that they would form a cross-species receptor for binding of dSNAP. In other cross-species experiments, it was demonstrated that yeast SNAP was able to functionally substitute for mammalian SNAP, presumably through interaction with SNARE proteins (28). We reasoned that since mSNAP has a higher amino acid identity with dSNAP (41), as it does to yeast SNAP, it would be likely that dSNAP would interact with mammalian SNAREs. The mammalian ternary complex, however, would prove to be an unsuitable substrate for testing the function of dNSFs since dSNAP did not bind mammalian syntaxin. This result suggests that despite the high identity between dSNAP and mSNAP, their properties have diverged in their interaction with syntaxin.

The biochemical properties of Drosophila SNAREs, however, appear to be conserved as they are able to form SDS-resistant complexes (fig. 3). Complexes appear more prominent in fractions in which membranes have been isolated, since relatively higher concentrations of dsyntaxin are present. These complexes appeared as multimers of 70 kDa, suggesting that multiple complexes of dsyntaxin, dVAMP and SNAP-25 are able to form, all of which are sensitive to heat treatment at 100 oC. Similar observations were made with mammalian lysates when SNAREs on synaptic vesicle were demonstrated to be disassembled in the presence of SNAP and NSF (20). The resemblance of the Western blot profile of dsyntaxin to the mammalian SNAREs, suggest that a measure of decrease or increase in complex formation could be used as an assay to characterize the function of dNSFs.

To characterize the function of dNSF-1, we employed the use of a known temperature sensitive paralytic mutation, called comatose TP7, that maps to the dNSF-1 locus. We discovered that, similar to other previously described temperature sensitive comatose alleles (38), the TP7 allele contains a single missense mutation in the D1 domain of dNSF-1. The mutation results in the conversion of a conserved proline residue at position number 397 (fig. 5) to serine. Since the D1 domain of NSF is known to be involved in the ATPase activity, it is possible that at non-permissive temperatures, this mutation may inhibit its activity (3, 45), rendering dNSF-1 non-functional. At this state, one would predict that SDS-resistant complexes would accumulate.

Surprisingly, heat shock treatment of comatose flies resulted in no detectable increase in SDS resistant complex formation compared to non-heat shocked or wild type flies, despite causing paralysis. A number of possibilities may explain why equivalent amounts of 7S complex are present in comatose flies whether or not they are heat shock treated. Some possibilities arise at the detection level. Given the fact that complexes assemble spontaneously, it is possible that during the homogenization steps, syntaxin proteins in wild-type samples may have been able to form 7S complexes equivalent to the heat shock treated comatose. However, this should have been minimized by the addition of 0.5% SDS, an agent that will prevent interaction of monomeric SNAREs to each other and therefore would reduce the probability of their interaction (20). Alternatively, one may not be able to directly correlate paralysis in comatose mutants with increases in SDS-resistant complexes in the whole organism, since these increases may be only localized and subtle, and therefore not detectable by Western blots. However, this problem may be averted by longer incubation periods at 38 oC of comatose flies as it may result in a much larger accumulation of ternary complexes compared to that of the wild type flies or control comatose flies.

Other possibilities originate at the functional level. The fact that two distinct NSFs are present in Drosophila and the inability of dNSF-2 to compensate for dNSF-1 mutation in comatose lines suggests that they have different functions. It is therefore conceivable that dNSF-1 and -2 each interact with a specific set of SNAREs, and dsyntaxin, the SNARE that we tested here, may be the substrate for dNSF-2 but not dNSF-1. Unfortunately, no mutants of dNSF-2 have been characterized to date and no other neuronal syntaxin homolog has been identified. Another possible scenario could be that the functions of dNSF-1 and -2 have diverged. Considering that mammalian NSF may have two different functions, one of uncoupling SNARE complexes (5) and second in creating a conformational change in the AMPA receptor GluR2 (46) , it is possible that each dNSF has become specialized to perform one of these two functions.

Future experiments will address these questions by expression and purification of wild type and mutant recombinant dNSF proteins and examining their effect on formation and disassembly of the 7S complexes present in fly lysates or formed by recombinant dSNAREs.

Back to the top.

<= Results DISCUSSION & CONCLUSSIONS References =>

| Discussion Board | Next Page | Your Symposium |
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