The neuralized homology repeat 1 domain of Drosophila neuralized mediates nuclear envelope association and delta-dependent inhibition of nuclear import.
Signaling by the Notch (N) pathway is critical for many developmental processes and requires complex trafficking of both the N receptor and its transmembrane ligands, Delta (Dl) and Serrate. neuralized encodes an E3 ubiquitin ligase required for N ligand internalization. Neuralized (Neur) is conserved from worms to humans and comprises two Neur homology repeat (NHR) domains, NHR1 and NHR2, and a carboxyl-terminal RING domain. We have previously shown that the RING domain is required for ubiquitin ligase activity and that NHR1 mediates binding to Dl, a ubiquitination target. In Drosophila, Neur associates with the plasma membrane and hepatocyte responsive serum phosphoprotein-positive endosomes. Here we demonstrate that Neur also exhibits nuclear envelope localization. We have determined that Neur subcellular localization is regulated by nuclear trafficking and that inhibition of chromosome region maintenance 1, a nuclear export receptor, interferes with Neur nuclear export, trapping Neur in the nucleus. Moreover, we demonstrate that nuclear envelope localization is mediated by the Neur NHR1 domain. Interestingly, Dl expression in Schneider cells is sufficient to inhibit Neur nuclear import and inhibition occurs in an NHR1-dependent manner, suggesting that Neur nuclear localization occurs in contexts where Dl expression is either low or absent. Consistent with this, we found that Neur exhibits nuclear trafficking and associates with the nuclear envelope in the secretory cells of the larval salivary gland and that overexpression of Dl can reduce Neur localization to the nucleus. Altogether, our data demonstrate that Neur localizes to the nuclear envelope and that this localization can be negatively regulated by Dl, suggesting a possible nuclear function for Neur in Drosophila.
Commisso
Cosimo
C
Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada.
Boulianne
Gabrielle L
GL
D021581:Active Transport, Cell Nucleus; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila / Q000737:chemistry* / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D028884:Karyopherins / Q000037:antagonists & inhibitors*; D007814:Larva / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism*; D010957:Plasmids; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors*; D051880:Receptors, Notch / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics; D000097606:Exportin 1 Protein
D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D018698:Glutamic Acid; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D028884:Karyopherins; D008565:Membrane Proteins; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D051880:Receptors, Notch; D012694:Serine; D044767:Ubiquitin-Protein Ligases; D000097606:Exportin 1 Protein; C064045:delta protein; C007293:DAPI; C071006:neur protein, Drosophila