Abstract
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.
Highlights
The ubiquitin/proteasome pathway influences a wide range of cellular processes, including mitosis, protein processing and quality control, response to infection, and programmed cell death [1,2,3]
Expression of Morgue mutants lacking the F box and Ubiquitin E2 Conjugase Variant (UEV) domain (MorgueDFB-UEV) or the zinc finger and F box (MorgueDZF-FB) exhibited reduced lethality compared to native, full length Morgue (Table 2). These results indicate that all three of the conserved Morgue domains, the zinc finger, F box, and UEV domain contribute to the lethal phenotype associated with Morgue over-expression phenotype, as deleting any of these domains attenuates the ability of Morgue to induce lethality
The Morgue over-expression phenotype exhibits dosage-sensitivity as animals heterozygous for P[da-gal4], P[UASMorgue] are fully viable while animals homozygous for one and heterozygous for the other P element exhibit extensive but generally incomplete lethality. This suggests that the lethal phenotype results only after a specific threshold level of Morgue expression is attained. This lethal phenotype provided a useful assay for Morgue activity and we utilized it to dissect the importance of the distinct domains of Morgue protein, as well as the conserved Gly421 active site residue
Summary
The ubiquitin/proteasome pathway influences a wide range of cellular processes, including mitosis, protein processing and quality control, response to infection, and programmed cell death [1,2,3]. Modification of proteins by ubiquitin involves the actions of a conserved enzymatic cascade, generally consisting of an E1 activator, E2 conjugase, and E3 ligase. Each of these enzymes is characterized by the presence of highly conserved domains that possess distinctive functional properties. E2s are typically small proteins that contain a ,130 amino acid conjugase domain This domain is characterized by the presence of several residues that interact with ubiquitin and an active site Cysteine residue that forms a covalent thioester linkage to the COOH-terminal Glycine residue of ubiquitin. Active E2s typically promote target protein ubiquitination by acting in conjunction with an E3 ligase. Structural studies have revealed the necessity for a precise arrangement and spacing of the SCF complex subunits to permit addition of ubiquitin moieties to specific substrates [16]
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