TRENDS in Neurosciences Vol.24 No.11 November 2001 http://tins.trends.com 0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01927-5 625 Research Update Research Update Research Update Research Update 15 Sonders, M.S. et al. (1997) Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants. J. Neurosci. 17, 960–974 16 Xia, J. et al. (1999) Clustering of AMPA receptors by the synaptic domain-containing protein PICK1. Neuron 22, 179–187 17 Otis, T.S. et al. (1997) Postsynaptic glutamate transport at the climbing fiber-Purkinje cell synapse. Science 277, 1515–1518 18 Auger, C. and Attwell, D. (2000) Fast removal of synaptic glutamate by postsynaptic transporters. Neuron 28, 547–558 19 Iversen, L. (2000) Neurotransmitter transporters: fruitful targets for CNS drug discovery. Mol. Psychiatry 5, 357–362 20 Amara, S.G. and Sonders, M.S. (1998) Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend. 51, 87–96 Scott L. Deken Matthew L. Beckman Michael W. Quick * Dept of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0021, USA. *e-mail: quick@nrc.uab.edu shibire’s enhancer is cancer’s suppressor David Deitcher Reliable neurotransmitter release requires an adequate pool of synaptic vesicles. These pools are maintained by the process of endocytosis. A recent paper revealed that dynamin,a GTPase essential for endocytosis, is regulated by Abnormal wing disc (Awd),a nucleoside diphosphate kinase which converts nucleoside diphosphates to nucleoside triphosphates. This finding suggests that access to GTP directly regulates endocytosis. In addition, awd is homologous to the tumor suppressor gene nm23, further suggesting that dynamin might be involved in tumorigenesis. Neurotransmitter release results from the fusion of synaptic vesicles at active zones. Following neurotransmitter release, the fused synaptic vesicle membrane is quickly retrieved to generate new synaptic vesicles. The rapid rate of endocytosis results from a coordinated effort of many proteins including clathrin, AP2, AP180, epsin, endophilin, amphiphysin, and dynamin. The lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] also seems integral for the process of endocytosis 1 . Release of the budding vesicle from the plasma membrane is dependent on the GTPase dynamin 2 . Dynamin in endocytosis The Drosophila mutant shibire (shi) results from mutations in the dynamin gene 3 . shi has pleiotropic effects but the most striking one is that of temperature- sensitive paralysis 4 . Heating shi flies causes paralysis by blocking endocytosis and preventing the resupply of synaptic vesicles. Electron microscopy studies on shi nerve terminals revealed a depletion of synaptic vesicles and the accumulation of pits in the plasma membrane surrounded by electron dense collars 5 , indicative of a trapped endocytic intermediate. From the analysis of shi, it is clear that dynamin plays a crucial role in endocytosis. But what regulates dynamin activity? Several proteins that interact with dynamin have been identified biochemically 6 . An alternative approach to identifying dynamin interactors is to use a genetic screen. abnormal wing disc is a shi enhancer In a recent report, an enhancer of shi was identified 7 . Enhancers are mutations in other genes that increase the severity of the original phenotype of the mutant. In this case, an enhancer of shi caused shi flies to paralyze at a lower temperature than normal. Enhancers usually act in the same biochemical pathway as the original mutant. Three of the enhancers recovered from the screen mapped to the gene abnormal wing disc (awd). awd is homologous to a putative tumor suppressor gene nm23 that functions as a nucleoside diphosphate kinase (NDK) 8 . NDKs convert nucleoside diphosphates (NDPs) to nucleoside triphosphates (NTPs). A connection between NDK and dynamin was unexpected because biochemical evidence had not previously implicated NDKs in the endocytosis pathway. Is the genetic interaction between awd and shi specific? Enhancer screens can identify specific interactors or nonspecific genes that just make a mutant line sicker. However, several lines of evidence support the specificity of the awd–shi genetic interaction. (1) Three alleles of awd were recovered from the screen. If the enhancers were nonspecific then it is probable that single alleles of several unrelated genes would have been identified instead of three alleles of the same gene. (2) An enhancer and the original mutation probably act in concert within the same tissue. awd is expressed in the nervous system and shi acts in the nervous system to cause paralysis. (3) Heated awd flies have synaptic transmission and vesicle depletion phenotypes that are similar to that of shi, indicating that both mutations affect endocytosis. (4) All the alleles of awd recovered from the screen affect the kinase activity of Awd, suggesting that this (lack of) activity is involved in the enhancement of the shi phenotype. awd could act as a guanine exchange factor for dynamin How might these proteins interact? Krishnan et al. 7 propose that either Awd could act as a local supply of GTP or deliver GTP as a guanine exchange factor (GEF). Thus, in awd mutants GTP loading of dynamin would be reduced, resulting in an endocytosis defect (Fig. 1). However, evidence for a direct interaction between the two proteins is lacking. Attempts at co-immunoprecipitation, co-immunolocalization, glutathione S-transferase-pulldown assays, and cell fractionation failed to show any association between Awd and dynamin. Although no physical interaction was observed, the idea that Awd provides GTP to dynamin is the most straightforward connection between the two proteins. It is possible that Awd does not directly interact with dynamin but utilizes another protein. Awd might act through PtdIns(4,5)P 2 NDKs can generate any NTP from ATP and NDPs (Ref. 9). Another hypothesis to