Abstract
Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N terminus and Middle). Although dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, MS analysis was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX, and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. Here, we demonstrate direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro-rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared with dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a post-synaptic-specific regulation of SH3PX1 by dNedd4Lo.
Highlights
Drosophila Nedd[4] is a HECT E3 ubiquitin ligase present in two major isoforms: short and long, with the latter containing two unique regions (N terminus and Middle)
We previously identified an interaction between dNedd4LoMid and SH3PX1 using mass spectrometry (MS) (Fig. 1A) (18)
Drosophila Schneider 2 (S2) cells were transiently co-transfected with HA-SH3PX1 and FLAG-dNedd4Lo or -dNedd4S, FLAGdNedd[4] immunoprecipitated (IP) and the IP immunoblotted for HA-SH3PX1
Summary
We previously identified an interaction between dNedd4LoMid and SH3PX1 using mass spectrometry (MS) (Fig. 1A) (18). Immunoblotting with anti-HA antibody showed that SH3PX1 binds only to the Mid region of dNedd4Lo in vitro and not the N-terminal region or GST alone (Fig. 1, C and D). Together, these binding experiments validate the MS results. We first generated mutant SH3PX1 lacking its SH3 domain (SH3PX1⌬SH3) This construct was transiently transfected into S2 cells along with FLAG-dNedd4Lo, and subjected to co-IP. We found that whereas the full-length SH3PX1-WT was able to bind the dNedd4LoMid region in vitro, this binding was abolished in the SH3PX1⌬SH3 mutant (Fig. 2, C and D) This indicates that SH3PX1 binds directly to the dNedd4Lo unique Mid region via its SH3 domain.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
