Regulation of SH3PX1 by dNedd4-long at the Drosophila neuromuscular junction.

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.

Regulation of Commissureless by the ubiquitin ligase DNedd4 is required for neuromuscular synaptogenesis in Drosophila melanogaster.

Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. We investigated whether dNedd4 and ubiquitination mediate this process. Here we show that Comm is expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm(2PY-->AY)], or bearing Lys-->Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm(10K-->R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY-->AY) and Comm(10K-->R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm(2PY-->AY)-monoUb or Comm(10K-->R)-monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis.

Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation.

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.