Liza M Mohanty et al. (2005) International Worm Meeting "Regulation of synaptobrevin (SNB-1) trafficking by the clathrin adaptor protein UNC-11 (C. elegans AP180)"

Aixa Alfonso, Liza M Mohanty

[International Worm Meeting, 2005]

UNC-11, the C. elegans homolog of AP180, is a monomeric clathrin adaptor protein known to function at the plasma membrane in the recycling of synaptic vesicles. UNC-11 also has a distinct role in the trafficking of the synaptic vesicle protein synaptobrevin (SNB-1). In the absence of functional UNC-11, SNB-1 is mislocalized to the commissures of motor neurons and to the dendrites of bipolar neurons. Normally commissures and dendrites are devoid of synaptic vesicles, and thus SNB-1 is not expected to be targeted to these locations. SNB-1 is the only synaptic vesicle protein known to be mislocalized in an unc-11 mutant background, indicating that the defect is not due to a general problem in synaptic vesicle recycling. Our goal is to determine how UNC-11 modulates SNB-1 trafficking in neurons.We propose three different models for UNC-11 function in SNB-1 trafficking. First, UNC-11 may retrieve SNB-1 from the axonal plasma membrane following exocytosis, thereby preventing lateral diffusion of SNB-1 to non-axonal sites. Alternatively, UNC-11 may function at the trans-Golgi network or endosome to correctly sort SNB-1 to a distinct transport vesicle class. Finally, UNC-11 may serve in the specific retrieval of SNB-1 from the plasma membrane following random delivery to non-axonal compartments such as the dendrite and cell body.We are currently investigating whether the interaction between UNC-11 and SNB-1 is direct or indirect. Previously we identified the early endosomal protein PQN-9 (C. elegans Hrs) as a binding partner of UNC-11. Mouse, fly, and yeast hrs mutants are defective in endocytic trafficking (Raiborg and Stenmark, 2002). We are now looking into the possibility of PQN-9 as an intermediary binding partner between UNC-11 and SNB-1. An interaction between PQN-9/Hrs and SNB-1 would suggest that the synaptobrevin trafficking defect occurs at the level of the early endosome. Additionally it would imply a novel role for UNC-11 at the early endosome in the correct sorting of SNB-1. Hrs is known to bind ubiquitin and sort ubiquitinated cargo proteins for trafficking to the lysosome (Raiborg et al., 2002). However, we have not observed direct ubiquitination of SNB-1. (This work was supported by the Department of Biological Sciences, University of Illinois at Chicago.)

Andrea M Holgado et al. (2001) International C. elegans Meeting "The C. elegans homologue of vertebrate EAST (C34G6.7) interacts with the C. elegans homologue of mammalian Hrs/Hrs-2 (C07G1.5), and the clathrin adaptor protein UNC-11A. Is there a link between clathrin mediated retrieval and synaptobrevin trafficking through the endocytic pathway? II. Characterization of Ce EAST expression."

Julie E Karr, Liza Mohanty, Andrea M Holgado, Aixa Alfonso

[International C. elegans Meeting, 2001]

Hrs and EAST are endosomal-associated proteins that are tyrosine phosphorylated in cells stimulated by growth factors such as EGF, HGF and PDGF. Both Hrs and EAST/Hbp have proposed roles in exocytosis as well as endocytosis (Bean et al., 2000; Murai and Kitamura, 2000). EAST is an avian protein that was initially identified as an EGF Receptor substrate and later was found to bind Eps15, a protein implicated in EGFR endocytosis. The rat homologue of EAST, Hbp, was found to bind Hrs via coiled-coil domains (reviewed in Komada and Kitamura, 2001). Since Hrs and UNC-11 also bind Eps15 (Ce Ehs-1 is homologous to mammalian Eps15), UNC-11, Ce Hrs and Ce EAST are likely to have a role in the trafficking of proteins from the plasma membrane through the endocytic pathway. In a yeast two-hybrid assay the nematode proteins C07G1.7 (Ce Hrs) and C34G6.7 (Ce EAST) bind to each of three distinct UNC-11 isoforms with varying strengths. Furthermore, the interaction is direct, as we are able to co-immunoprecipitate either protein with UNC-11 using in vitro synthesized proteins. Although we have been unable to co-immunoprecipitate from nematode extracts endogenous UNC-11 with either of these proteins, we have documented an in vivo interaction between endogenous Ce Hrs and Ce EAST. To characterize the role of Ce EAST in the endocytic trafficking we have characterized the pattern of expression of the protein encoded by the C34G6.7 open reading frame. We have generated antibodies against a recombinant, full-length, GST-fusion protein. A single protein of approximately 57 kDa is detectable in protein extracts using affinity-purified antibodies. Immunohistochemical analysis of whole worms has revealed that Ce EAST, like Ce Hrs, is ubiquitously expressed and is detected intracellularly in a punctate pattern that is reminiscent of endosomes. The pattern of expression is also dynamic and detectable throughout development. In the adult, Ce EAST, like Ce Hrs, is enriched in the spermatheca, coelomocytes and the pharyngeal intestinal valve. Furthermore, Ce EAST immunostaining, like that of Ce Hrs, only partially colocalizes with the synaptic vesicle marker UNC-17 at the ventral and dorsal nerve cords. However, Ce EAST is more abundant than Ce Hrs in the cell bodies of the ventral cord motor neurons. To determine the function of Ce EAST 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. In contrast to Ce Hrs, Ce EAST is not essential for viability and development. The progeny of injected animals look and develop like wild type animals. Since RNAi is known to be ineffective in inactivating the function of proteins in the nervous system we are in the process of isolating a loss of function mutant in Ce EAST to determine whether it would affect trafficking of the synaptic vesicle protein synaptobrevin. This work has been supported by UIC departmental funds to AA and an NIH grant (NS32449) to AA.