Drosophila Neuralized Is a Ubiquitin Ligase that Promotes the Internalization and Degradation of Delta  Eric C. Lai, Gisèle A. Deblandre, Chris Kintner, Gerald M. Rubin  Developmental Cell  Volume 1, Issue 6, Pages 783-794 (December 2001) DOI: 10.1016/S1534-5807(01)00092-2

Figure 1 Phenotypes Produced by Misexpression of Neur and Neur RING Are Dominantly Suppressed by Mutation of UbcD1, which Encodes an E2 Ubiquitin-Conjugating Enzyme GMR-Gal4, UAS-neur (A), GMR-Gal4, UAS-neur; UbcD1S278/+ (B). Interommatidial bristle tufting induced by Neur misexpression is partially suppressed by UbcD1. Insets are higher magnification views that more clearly depict the suppression of bristle tufting. GMR-Gal4, UAS-neur RING (C), GMR-Gal4, UAS-neur RING; UbcD1S278/+ (D). Small eye phenotype induced by Neur RING misexpression is partially suppressed by lowering UbcD1 level. Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)

Figure 2 The Neur RING Finger Is a Ubiquitin Ligase Ubiquitination assays were performed using the indicated sources of E3 enzyme and subjected to Western blot analysis to detect ubiquitinated proteins (lanes 1–4) and GST fusion proteins (lanes 5–8). Ubiquitin-protein conjugates (bracket) are seen only in the presence of His-WWP-HECT (lane 2) or GST-Neur RING (lane 3), and not with GST (lane 1) or the mutant GST-Neur RING HC→AA (lane 4). In addition, GST-Neur RING can autoubiquitinate (lane 7) as seen by the ladder of α-GST-reacting proteins (bracket), while GST-Neur RING HC→AA cannot (lane 8). The sizes of unubiquitinated GST and GST-Neur RING are marked with arrows. Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)

Figure 3 Altered Subcellular Distribution and Steady-State Levels of Dl in neur Mutant Eyes (A–I) Third instar eye discs of the indicated genotypes were doubly labeled with Dl (red) and Neur (green). (A) In wild-type, Dl posterior to the morphogenetic furrow (mf) is found primarily in vesicles. (B) In the neur mutant disc, the level of Dl is elevated. (C) In the absence of E(spl)-C function, Dl is strongly reduced posterior to the morphogenetic furrow; some regions of the disc do not contain Dl (arrow). (D–F) Neur is present in a regular pattern in wild-type (D), is largely absent in the neur mutant disc (E), and is broadly expressed in the E(spl)-C mutant disc (F). (G–I) Defect in Dl localization is seen more clearly in higher magnification confocal images from rows ∼6–12 of the eye disc. Only in the absence of neur function is Dl found significantly at the plasma membrane (H). (J–L) Wing disc containing a neurIF65 mutant clone that overlaps proneural clusters of the anterior wing margin ([K and L], asterisks); the clone is marked by the absence of GFP (J), and the disc is stained for Dl (K and L). Two focal planes are shown, depicting basal, vesicular Dl (K) and apical, plasma membrane Dl (L). Dl is not significantly altered in the clone. Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)

Figure 4 Neur Reduces the Steady-State Levels of Dl, an Activity that Requires Its RING Finger and the 26S Proteasome (A–C) dpp-Gal4>UAS-nuclear GFP disc stained for Dl; GFP (A), Dl protein (B), and merge (C). The domain of dpp-Gal4 activity overlaps the developing L3 wing vein ([B], arrow) and abuts the L4 wing vein (arrowhead). (D–I) Dl-stained wing discs (D–F) and adult wings (G–I) from dpp-Gal4>2xUAS-neur (D and G), dpp-Gal4>UAS-neurΔRF (E and H), and dpp-Gal4;>2xUAS-neur, UAS-neurΔRF (F and I). Misexpression of Neur leads to a decrease in L3 Dl protein in the wing disc ([D], arrow) and loss of campaniform sensilla in the adult wing ([G], bracket); inset depicts a portion of the bracketed region that would normally contain a single campaniform sensilla. Misexpression of NeurΔRF increases the level of Dl on L3 ([E], arrow); corresponding adult wings display many ectopic campaniform sensilla ([H], bracket and inset), a mildly thickened L3 wing vein, and a notched wing margin (arrow). Mutual suppression of Neur and NeurΔRF effects on Dl levels (F) and adult wing phenotypes (I), with only a small number of ectopic campaniform sensilla ([I], inset). (J) Prolonged reduction of proteasome activity increases the steady-state level of Dl (bracket); disc is from a bxMS1096-Gal4/X; UAS-DTS5 animal that was raised at 29°C and stained for Dl. (K and L) Regulation of Dl by Neur requires the proteasome. dpp-Gal4>2xUAS-neu, UAS-DTS5 animals were raised at 22°C, then shifted to 29°C for 3 hr prior to dissection and staining for Dl. Vesicular Dl is observed along L3 (within the Neur-expressing domain) while Dl is mostly localized at the plasma membrane along L4. Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)

Figure 5 Neur-Mediated Degradation of Newly Synthesized Delta Requires the RING Finger Third instar wing imaginal discs were stained for Dl (red) or Neur (green); merged images are shown in (F), (I), and (L). All images are taken from the central dpp-Gal4-expressing domain of the wing pouch. With the exception of (B) and (C), discs were heat-shocked at 37°C for 40 min and allowed to recover at 22°C for the indicated periods of time before dissection. Insets depict higher magnification views of each respective panel. (A) hs-Dl/+: 40 min recovery. A high level of Dl is ubiquitously induced upon heat shock and Dl is initially associated with the apical plasma membrane. (B) dpp-Gal4>2xUAS-neur; Neur is largely associated with the plasma membrane, although some punctate, apparently vesicular, staining is also observed. (C) Wild-type disc stained for Neur; a low level of Neur is detected in sensory organ precursors of the anterior wing margin, and it is associated with the plasma membrane. (D–I) dpp-Gal4>2xUAS-neur; hs-Dl/+. (D–F) 40 min recovery. (D) In the presence of Neur, Dl is rapidly targeted to vesicles (bracket). (E) Neur is also internalized at this time point, although much of it remains at the plasma membrane. Colocalization of Neur and Dl (yellow particles) are seen clearly at higher magnification ([F], inset). (G–I) 90 min recovery. (G) Almost all of the induced Dl has been degraded within the Neur-expressing domain (bracket). (H) At this time, Neur protein is found at both the plasma membrane and the cytoplasm. (J–L) dpp-Gal4>UAS-neurΔRF; hs-Dl/+: 120 min recovery. In the presence of NeurΔRF, Dl is stabilized in large apical vesicular structures ([J], inset), with a lower amount seen at the apical plasma membrane. NeurΔRF shows partial colocalization with Dl in apical vesicular structures ([K and L], insets), but is primarily associated with the plasma membrane. It also accumulates to higher levels than does Neur (compare with [B], [E], and [H]). Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)

Figure 6 Neur and NeurΔRF Are Coimmunoprecipitated (Co-IPed) with Dl (A and B) HEK-293 cells were transfected with plasmids encoding the indicated proteins. Total cell lysates (lanes 1–4) or polyoma-immunoprecipitated material (lanes 5–9) was sequentially probed for Myc-tagged proteins (A) and, following stripping of antibodies, polyoma-tagged proteins (B). Neur and NeurΔRF (ΔRF) typically run as two to three bands in the ∼100–120 kDa range (lanes 1 and 2), ∼10–20 kDa larger than their predicted molecular weights. The size differences between Neur and NeurΔRF (which are 5 kDa smaller) are not well resolved in these gels. Note that NeurΔRF accumulates to a higher level than does Neur (compare lanes 1 and 2). Neur (lane 5) and NeurΔRF (lane 6) are co-IPed with Dl. NeurΔNHR1 (ΔNHR1) (lanes 3 and 7) and LRP-C (lanes 4 and 8) do not associate with Dl, and NeurΔRF is not significantly co-IPed with control polyoma-tagged GFP (lane 9); HC, heavy chain. (B) Subsequent reprobing of these blots shows that equivalent amounts of Dl or GFP were IP-ed in each lane. A fragment corresponding in size to the intracellular domain of Dl (DlIC) is found in all cell lysates, independent of the cotransfected plasmid; DlIC in lane 8 was cut off the bottom of the gel (asterisk). (C) In vivo association of Dl and Neur/NeurΔRF. Total lysates (lanes 1–3) and Dl-immunoprecipitated proteins (lanes 4–7) from imaginal disc and brain complexes were isolated from white (lane 4), sca-Gal4>UAS-myc-sty (lanes 1 and 5), sca-Gal4>UAS-myc-neur (lanes 2 and 6), and sca-Gal4>UAS-myc-neurΔRF (lanes 3 and 7) larvae and probed for Myc-containing proteins. Note that a Myc-crossreacting band (lane 4, asterisk) similar in size to one of the prominent Neur/ΔRF bands is present in Dl immunoprecipitates from white and Sty-expressing tissue, although no Sty protein is associated with Dl (lane 5). A small amount of slower migrating Neur is detected above this crossreacting band (lane 6), while a much larger amount of NeurΔRF is co-IPed with Dl (lane 7). Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)

Figure 7 Model for Neur Function Neur is composed of two NHR domains (blue ovals), which appear to mediate association with the plasma membrane and recognition of Dl, and a C-terminal RING finger (red), which is a ubiquitin ligase domain. Neur may ubiquitinate Dl (green) at the plasma membrane, promoting endocytosis of a Neur/Dl-containing complex. Internalized Dl is rapidly degraded by a mechanism which likely involves both the proteasome and lysosome. Neur-mediated reduction of cell surface Dl increases the ability of that cell to activate the N receptor (yellow) and allow target gene transcription by the nuclear NIC/Su(H) complex. Developmental Cell 2001 1, 783-794DOI: (10.1016/S1534-5807(01)00092-2)