An antifreeze protein (AFP) from a midge (Chironomidae) was recently discovered and modelled as a tightly wound disulfide-braced solenoid with a surface-exposed rank of stacked tyrosines. New isoforms of the midge AFP have been identified from RT-PCR and are fully consistent with the model. Although they differ in the number of 10-residue coils, the row of tyrosines that form the putative ice-binding site is conserved. Recombinant midge AFP has been produced, and the properly folded form purified by ice affinity. This monomeric AFP has a distinct circular dichroism spectrum, a melting temperature between 35 and 50°C and is fully renaturable on cooling. Mutagenesis of the middle tyrosine in the rank of seven eliminates antifreeze activity, whereas mutation of a tyrosine off this predicted icebinding face had no such effect. This AFP has unusual properties compared to other known AFPs. First, its freezing-point depression activity is intermediate between that of the hyperactive and moderately active AFPs. As with hyperactive AFPs, when midge AFP-bound ice crystals exceed their freezing-point depression, ice grows explosively perpendicular to the c-axis. However, midge AFP does not bind to the basal plane of ice as do hyperactive AFPs, but rather to a pyramidal plane that is at a shallower angle relative to the basal plane than binding planes of moderate AFPs. These properties distinguish midge AFP from all other ice-binding proteins and the intermediate activity level fits well to the modest challenge of protecting newly emerged adult insects from late spring frosts. DatabaseNucleotide sequences of new midge AFP isoforms are available in the GenBank database under accession numbers KU094814-8. Sequences will be released after publication.

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 postsynaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo postsynaptically 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 postsynaptic-specific regulation of SH3PX1 by dNedd4Lo.