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Updated: May 7, 2026
Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments
Published on: May 30, 2017
Drosophila morgue associates with SkpA and polyubiquitin in vivo
Ying Zhou1, Yiqin Wang, Barbara A Schreader
1Department of Biology, University of Massachusetts, Amherst, Massachusetts, United States of America ; Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida, United States of America.
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.
Insights
Morgue protein over-expression causes lethality in Drosophila, revealing its role in programmed cell death. Key domains are crucial for its function, and it interacts with SkpA and poly-ubiquitin, suggesting a role in proteasomal pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Morgue is a Drosophila protein involved in programmed cell death and circadian rhythms.
- Over-expression of wild-type Morgue leads to organismal lethality, providing a basis for functional studies.
Purpose of the Study:
- To investigate the functional importance of specific Morgue domains (zinc finger, F-box, UEV, active site Glycine) using an in vivo lethality assay.
- To identify Morgue-associated proteins and elucidate its molecular interactions and cellular functions.
Main Methods:
- In vivo functional assay based on Morgue over-expression-induced lethality.
- Site-directed mutagenesis to assess the role of specific Morgue domains.
- Affinity purification of Flag:Morgue followed by mass spectrometry to identify interacting proteins.
- Analysis of poly-ubiquitin linkage topology (Lys-48).
Main Results:
- Removal of the zinc finger or UEV domain reduced Morgue's lethality-inducing and cell death-enhancing effects.
- The F-box and active site Glycine were not essential for Morgue-induced lethality or cell death enhancement.
- SkpA and a Lys-48 linked ubiquitin multimer were identified as Morgue-associated proteins.
- Specific regions of Morgue are required for poly-ubiquitin binding.
Conclusions:
- Morgue functions as a multi-functional ubiquitin-binding protein.
- Its zinc finger and UEV domains are critical for inducing lethality and enhancing cell death.
- Morgue likely acts within an SCF-type ubiquitin E3 ligase complex and plays a role in proteasome-mediated protein turnover via Lys-48 poly-ubiquitination.
