The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling

Abstract

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

“…Another possibility is that synaptic vesicles are not depleted because the NFP1 peptide causes a simultaneous inhibition of exocytosis (see next paragraph) that prevents vesicles from being depleted despite the inhibition of endocytosis. In contrast to our results, inhibition of Eps15 expression by RNA interference causes a massive depletion of synaptic vesicles in C. elegans (14). This severe phenotype may be caused by the fact that Eps15 interacts with a large number of endocytic proteins, and all of these interactions will be disrupted following loss of Eps15.…”

“…Furthermore, clathrin-mediated endocytosis is inhibited in cells that overexpress Eps15 mutants lacking either the DPF-rich AP-2 binding domain (16) or the EH domains (17). Genetic disruption of the Caenorhabditis elegans homologue of Eps15, EHS-1, results in an uncoordinated phenotype and depletes synaptic vesicles, supporting a role for EHS-1 in SV recycling (14). In addition, Pan1p, a yeast homologue of Eps15, binds to and activates the actin-nucleating Arp2-Arp3 complex.…”

“…Our work here demonstrates that Eps15 is indeed an important regulator of AP180-mediated clathrin assembly. Eps15 has also been implicated in vesicle scission because of its interactions with dynamin (14) and in uncoating by virtue of its interactions with synaptojanin (8). By participating in cytoskeletal rearrangements, cargo selection, clathrin-coated pit formation, clathrincoated vesicle scission, and clathrin-coated vesicle uncoating, Eps15 may direct the temporal linkage of these sequential reactions that underlie endocytosis.…”

“…This DPF-rich domain was identified as one of the first of many binding partners that interact with the ␣-ear domain of AP-2, another clathrin assembly protein (10 -13). Although its exact site of interaction is still unknown, Eps15 also interacts both biochemically and genetically with dynamin, a GTPase believed to catalyze the fission reaction as nascent coated vesicles are released from the membrane (14). Given that many of the binding partners of Eps15 are associated with clathrin, it is likely that Eps15 might participate in some aspect of clathrin-mediated endocytosis.…”

Synaptic tetraspan vesicle membrane proteins are conserved but not needed for synaptogenesis and neuronal function in Caenorhabditis elegans

“…5D). Some synaptic vesicle proteins are required to maintain normal transmission in response to extreme temperatures (25,26). To examine whether there was an underlying temperature sensitivity, worms were grown for several generations either at 15°C or at 25°C and then subjected to aldicarb testing at the respective temperatures.…”