The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Stonin 2 is a mammalian synaptic protein that is thought to act as an adaptor to recruit the synaptic vesicle protein synaptotagmin to sites of synaptic vesicle endocytosis (the Drosophila stonin ortholog is STNB). To characterize the role of stonins in synaptic vesicle endocytosis, we examined unc-41 mutants; the unc-41 locus encodes the stonin/STNB ortholog in C. elegans. The unc-41 gene encodes two protein isoforms that are differentially expressed in the C. elegans nervous system: UNC-41A is expressed in all neurons, while UNC-41B is expressed in a subset of neurons, including the GABAergic motorneurons in the ventral nerve cord. The UNC-41 proteins are localized to synapses, similar to the pattern seen with SNT-1. We examined the synaptic localization of GFP-tagged SNT-1 in unc-41 mutants. Consistent with studies in Drosophila, tagged synaptotagmin is dimmer and mislocalized in unc-41 mutants, and fluorescence is also observed in non-synaptic regions such as commissures. In contrast, GFP::UNC-41 is properly localized in snt-1 mutants, demonstrating a unidirectional relationship for localization. Ultrastructural analysis indicates that unc-41 mutants exhibit a defect in membrane retrieval: synaptic vesicle numbers are reduced by 50% compared to wild type. We conclude that UNC-41 is required to recruit synaptotagmin and recover membrane during synaptic vesicle endocytosis. apm-2 mutants (pka dpy-23), lacking the m2 subunit of AP2, exhibit a similar deficit in synaptic vesicle endocytosis. Because in mammals, both m2 and stonin 2 can bind synaptotagmin, we considered the possibility that these proteins carried out overlapping functions in C. elegans. However, nematodes lacking both UNC-41 and m2 (unc-41 apm-2 double mutants) exhibit a decrease in synaptic vesicle number comparable to either single mutant. Our data suggest that UNC-41 and m2 function in the same pathway for synaptic vesicle membrane recycling. (Supported by NIH grants NS034307 to E.J. and NS033187 and GM059642 to J.R.).

Authors: Mullen, Greg, Grundahl, Kiely, Gu, Mingyu, Watanabe, Shigeki, Hobson, Robert, McManus, John, Mathews, Ellie, Jorgensen, Erik, Rand, Jim

Affiliations:
- Gen Models Disease Res Program, Oklahoma Med Res Foundation, Oklahoma City, OK.
- Department of Biology, University of Utah, Salt Lake City, Utah