Drosophila Morgue is an F box/ubiquitin conjugase domain protein important for grim-reaper mediated apoptosis.

In Drosophila melanogaster, apoptosis is controlled by the integrated actions of the Grim-Reaper (Grim-Rpr) and Drosophila Inhibitor of Apoptosis (DIAP) proteins (reviewed in refs 1 4). The anti-apoptotic DIAPs bind to caspases and inhibit their proteolytic activities. DIAPs also bind to Grim-Rpr proteins, an interaction that promotes caspase activity and the initiation of apoptosis. Using a genetic modifier screen, we identified four enhancers of grim-reaper-induced apoptosis that all regulate ubiquitination processes: uba-1, skpA, fat facets (faf), and morgue. Strikingly, morgue encodes a unique protein that contains both an F box and a ubiquitin E2 conjugase domain that lacks the active site Cys required for ubiquitin linkage. A reduction of morgue activity suppressed grim-reaper-induced cell death in Drosophila. In cultured cells, Morgue induced apoptosis that was suppressed by DIAP1. Targeted morgue expression downregulated DIAP1 levels in Drosophila tissue, and Morgue and Rpr together downregulated DIAP1 levels in cultured cells. Consistent with potential substrate binding functions in an SCF ubiquitin E3 ligase complex, Morgue exhibited F box-dependent association with SkpA and F box-independent association with DIAP1. Morgue may thus have a key function in apoptosis by targeting DIAP1 for ubiquitination and turnover.

The unique Morgue ubiquitination protein is conserved in a diverse but restricted set of invertebrates.

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways.

Drosophila sickle is a novel grim-reaper cell death activator.

The Drosophila genes reaper, head involution defective (hid), and grim all reside at 75C on chromosome three and encode related proteins that have crucial functions in programmed cell death (reviewed in ). In this report, we describe a novel grim-reaper gene, termed sickle, that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression was detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression was detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death was antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression did not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an r/grim chimera or reaper.

Drosophila morgue associates with SkpA and polyubiquitin in vivo.

Morgue is a unique ubiquitination protein that influences programmed cell death and circadian rhythms in Drosophila. We have found that over-expression of wild-type Morgue results in organismal lethality. This over-expression phenotype was used as the basis for an in vivo functional assay to investigate the importance of the Morgue zinc finger, F box, Ubiquitin E2 Conjugase Variant (UEV) domain, and active site Glycine residue. Removal of the zinc finger or UEV domain reduced Morgue's ability to induce lethality and enhance cell death. In contrast, lack of the F box as well as several different substitutions of the active site Glycine did not alter Morgue-induced lethality or cell death enhancement. To further characterize Morgue functions, a Flag:Morgue protein was used to isolate Morgue-associated proteins from whole adult Drosophila. Mass spectrometry analysis of the Morgue-associated proteins identified SkpA as well as a ubiquitin multimer. The identification of SkpA is consistent with previous in vitro studies and further suggests Morgue acts in an SCF-type ubiquitin E3 ligase complex. The identification of poly-ubiquitin was unexpected and this interaction had not been previously identified. The associated poly-ubiquitin was found to exhibit a Lys-48 topology, consistent with distinct functions of Morgue in proteasome-mediated protein turnover. Multiple regions of Morgue were subsequently shown to be required for poly-ubiquitin binding. Overall, Morgue is a novel multi-functional ubiquitin-binding protein.

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit.

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry.

Targeted expression of p35 reveals a role for caspases in formation of the adult abdominal cuticle in Drosophila.

Programmed cell death is a fundamental aspect of metazoan development associated with the elaboration of disparate tissues and structures. Specialized cysteine proteases, the caspases, are mediators of cell death; once activated they cleave substrate proteins to dismantle doomed cells. Caspase activity is regulated by several cellular and viral inhibitors. The baculovirus p35 protein blocks the action of a wide range of caspases and inhibits cell death in divergent species. Here, we utilize the Gal4/UAS system to target p35 expression and analyze the requirements of caspase activity for development in Drosophila. We confirm that cell death is essential for proper morphogenesis of the adult male external genitalia and distal portions of the legs. In addition, we find that caspases are also required for elimination of larval epidermal cells and normal elaboration of the adult abdominal cuticle by histoblast derivatives. In particular, rescued p35-expressing larval epidermal cells accumulate along the abdominal midline and are associated with corresponding splits in both dorsal and ventral cuticle structures. This study reveals a novel role for cell death in a specific morphogenetic processes.

Drosophila morgue influences cell numbers and positions in the embryonic nervous system.

Morgue is a unique multi-domain protein that contains a zinc finger motif, an F box, and a variant E2 conjugase domain. The presence of these domains suggests potentially complex and novel functions for Morgue in ubiquitination pathways. Morgue was originally identified via its gain-of-function enhancement of eye cell death phenotypes in Drosophila and ectopic expression of Morgue also influences circadian rhythms. However, there is as yet little known about Morgues normal developmental or physiological functions. To address this issue, we generated several morgue loss-of-function mutants via P element excision mutagenesis and analyzed the mutant phenotypes during the fly life cycle. These studies revealed that morgue null mutants are viable, though approximately 10% of the mutants exhibit defects in pupal spiracle eversion and malformations in the adult abdominal cuticle. In addition, a similar subset of morgue mutant embryos exhibited alterations in the normal number, position, or morphology of specific neurons and glia. Analysis of Morgue protein localization was addressed through generation of a transgenic fly strain that expresses a GFP::Morgue fusion protein. Use of this strain revealed Morgue protein localization in multiple cellular compartments, including nuclei, cytoplasm and membranes. Taken together, these diverse phenotypes and distribution patterns suggest pleiotropic functions for Morgue.

Drosophila Pelle phosphorylates Dichaete protein and influences its subcellular distribution in developing oocytes.

The Drosophila Dichaete gene encodes a member of the Sox family of high mobility group (HMG) domain proteins that have crucial gene regulatory functions in diverse developmental processes. The subcellular localization and transcriptional regulatory activities of Sox proteins can be regulated by several post-translational modifications. To identify genes that functionally interact with Dichaete, we undertook a genetic modifier screen based on a Dichaete gain-of-function phenotype in the adult eye. Mutations in several genes, including decapentaplegic, engrailed and pelle, behaved as dominant modifiers of this eye phenotype. Further analysis of pelle mutants revealed that loss of pelle function results in alterations in the distinctive cytoplasmic distribution of Dichaete protein within the developing oocyte, as well as defects in the elaboration of individual egg chambers. The death domain-containing region of the Pelle protein kinase was found to associate with both Dichaete and mouse Sox2 proteins, and Pelle can phosphorylate Dichaete protein in vitro. Overall, these findings reveal that maternal functions of pelle are essential for proper localization of Dichaete protein in the oocyte and normal egg chamber formation. Dichaete appears to be a novel phosphorylation substrate for Pelle and may function in a Pelle-dependent signaling pathway during oogenesis.

Maternal expression and function of the Drosophila sox gene Dichaete during oogenesis.

Members of the Sox family of DNA-binding HMG domain proteins have been shown to regulate gene transcription in a wide range of developmental processes, including sex determination, neurogenesis, and chondrogenesis. However, little is known about their potential functions in developing germline tissues. In Drosophila, the Sox protein Dichaete (a.k.a., Fish-hook) is a member of the SoxB subgroup whose HMG domain shares strong sequence similarity to that of vertebrate Sox2. Dichaete exhibits dynamic expression in embryonic and larval stages and has pleiotropic functions in a variety of tissues. In this study, we extend analyses of Dichaete function and show that expression of Dichaete protein is detected in the developing oocyte during early to mid stages of oogenesis. Strikingly, Dichaete exhibits cytoplasmic distribution and is not detected in the oocyte nucleus. Germline mosaic analyses revealed that the Dichaete gene has maternal functions that influence dorsal/ventral patterning of the egg chamber. Dichaete mutant eggs exhibit defects in formation of the dorsal appendages, differentiation of dorsal/anterior follicle cells, and mislocalization of Gurken protein and gurken mRNA. Dichaete protein was shown to possess RNA-binding capabilities, suggesting a direct post-transcriptional role in regulating RNA functions.

Drosophila morgue and the intersection between protein ubiquitination and programmed cell death.

In Drosophila, cell survival decisions are mediated by the integrated functions of the Grim-Reaper death activators and Inhibitor-of-Apoptosis-Proteins (IAPs), such as DIAP1, to regulate caspase activities. We recently identified a gene that enhances the actions of the Grim-Reaper proteins and negatively regulates the levels of DIAP1 protein. This gene, morgue, encodes a novel protein that contains both an F box and a ubiquitin conjugase domain. Interestingly, the Morgue conjugase domain lacks the active site cysteine required for covalent linkage to ubiquitin. Morgue could target IAPs and other proteins for ubiquitination and proteasome-dependent turnover by acting either in an SCF ubiquitin E3 ligase complex, or as a ubiquitin E2 conjugase enzyme variant (UEV) in conjunction with a catalytically active E2 conjugase. Morgue is evolutionarily conserved, as a Morgue ortholog was identified from the mosquito, Anopheles gambiae. Elucidation of morgue function should provide novel insights into the mechanisms of ubiquitination and programmed cell death.