Our general goal is to understand the mechanism(s) of synaptic vesicle (SV) recycling at the active zone. Specifically we want to address the contribution of clathrin-mediated endocytosis to this process. Our approach has been to characterize the synaptic transmission defects of mutants lacking the function of the monomeric clathrin assembly protein UNC-11. In the absence of
unc-11 function there is a generalized defect in neurotransmitter release probably as a result of a decrease in SV fusion. Ultrastructural analysis revealed an accumulation of vesicular structures that are larger than mature synaptic vesicles and the mislocalization of synaptobrevin to all neuronal membranes including those in pre- and post-synaptic compartments (Nonet et al., 1999, Krump et al., 2001). Thus, we are testing the hypothesis that UNC-11 has a role in the trafficking of synaptobrevin from the pre-synaptic plasma membrane through the endocytic pathway. Interestingly, a yeast two-hybrid screen designed to identify proteins that interact with UNC-11A (one of six possible isoforms derived from the gene
unc-11 ), identified two candidate-binding proteins which have been implicated in trafficking between endosomes and lysosomes, C07G1.5 and C34G6.7. The protein encoded by C07G1.5 is homologous to mammalian Hrs/Hrs-2, whereas the open reading frame C34G6.7 is homologous to the vertebrate proteins EAST/Hbp. Both Hrs ( H epatocyte growth factor r egulated tyrosine kinase s ubstrate) and EAST/Hbp ( E GFR A ssociated protein with S H3 and T AM domains/ H RS b inding p rotein) have been implicated in receptor-mediated endocytosis and promote internalization of proteins targeted to the recycling or degradation pathways (reviewed in Komara and Kitamura, 2001). To characterize the expression pattern of Ce Hrs in the nematode we have generated antibodies against a recombinant, full-length, GST-fusion protein. A single protein of approximately 105 kDa is detectable in protein extracts using affinity-purified antibodies. Immunohistochemical analysis of whole worms has revealed that Ce Hrs is expressed in all cells in an intracellular punctate pattern that is reminiscent of endosomes. The expression pattern is dynamic and detectable throughout development. In the adult, Ce Hrs is enriched in the spermatheca, coelomocytes and the pharyngeal intestinal valve. At the ventral and dorsal nerve cords, Ce Hrs immunostaining only partially colocalizes with the synaptic vesicle marker UNC-17. To determine the function of Ce Hrs in vivo we have used double stranded RNA interference (RNAi) to knock out the function of this protein in the progeny of the injected animals. The RNAi analysis indicates that Ce Hrs plays an important role in nematode development. About 82% of the RNAi treated progeny die at embryonic and larval stages. In addition, the surviving animals take three times longer to reach adulthood and in most cases develop phenotypes similar to those displayed by well-characterized mutants in signal transduction pathways. To analyze the function of Ce Hrs in the nervous system we are in the process of isolating deletion mutants and generating transgenic lines expressing dominant negative forms of the protein in the nervous system. This work has been supported by UIC departmental funds to AA and an NIH grant (NS32449) to AA.