Functional Characterization of Mul1, a Ubiquitin Ligase Involved in Mitochondrial Recycling
Location
Medical Education Building, LSUHSC-NO
Presentation Date
10-10-2019 10:00 AM
End Date
10-10-2019 12:00 PM
Description
Mitochondria play a crucial role in multiple cellular processes. Aside from supporting aerobic respiration, mitochondria are involved in steroid synthesis, calcium homeostasis and induction of apoptosis. They are highly dynamic organelles that undergo fission, fusion and distribute throughout the cell using the cytoskeletal network. Defects in mitochondrial dynamics can result in accumulation of dysfunctional mitochondria and enhanced oxidative stress, ultimately leading to neurodegenerative diseases such as Charcot-Marie-Tooth disease and autosomal dominant optic atrophy. Mul1 (Mulan) is a mitochondrial E3 ubiquitin ligase that functions in mitochondrial dynamics and integrity. Mul1 is expressed in all tissues, with the highest abundance in the heart, where mitochondrial quality control is particularly acute. Ectopic expression of Mul1 in HeLa cells leads to defects in both mitochondrial morphology and subcellular distribution. Mul1 localizes to the outer mitochondrial membrane and contains two transmembrane domains, and a C terminus that faces the cytosol. Mul1 exerts its regulatory effects by ubiquitination, which is a three-step process involving ubiquitin activation through E1 activating enzyme, transfer of a high energy ubiquitin thioester to E2 carrier protein to form E2~ubiquitin thioester and conjugation of E2-bound ubiquitin to target protein. The last step is catalyzed by E3 ubiquitin ligases. Previous work suggests that Mul1 specifically mediates conjugation of Mfn2 (mitofusin2) with ubiquitin. However, a recent retraction by Lokireddy et al. questions Mul1 conjugation of Mfn2. The aim of this study was to enzymatically characterize the mechanism of Mul1 ubiquitin conjugation. The cytoplasmic domain of Mul1 (accession number NP_078820, residues 262-352) was optimized for bacterial expression and subcloned into a pGEX expression vector. Recombinant GST-Mul1 was expressed in Escherichia coli BL21 (DE3) cells after induction with IPTG. The resulting protein was purified by affinity chromatography using glutathione sepharose and the GST was subsequently cleaved using thrombin. Mul1 was then resolved from unbound GST by affinity chromatography and used in subsequent experiments. Additionally, cytoplasmic domains of Mfn1 (accession number NM_033540, residues 1-580) and Mfn2 (residues 1-599, accession number NM_014874) were subcloned into pGEX, expressed and purified in a similar manner. A 125I-polyubiquitin chain formation functional assay was used to identify Ubc5A as a cognate E2 ubiquitin carrier for Mul1. Activity assays were also used to test whether recombinant mitofusin proteins can serve as substrates for Mul1-dependent conjugation with ubiquitin. Results showed that Mul1 shows broad linkage specificity in the assembly of free polyubiquitin chains in the absence of substrate. The assembly of unanchored ubiquitin chains was inhibited by Mfn2 but not by Mfn1, which suggests that the former binds Mul1 but does not serve as a substrate for ubiquitin conjugation.
Recommended Citation
Mirzalieva, Oygul, "Functional Characterization of Mul1, a Ubiquitin Ligase Involved in Mitochondrial
Recycling" (2019). Medical Student Research Poster Symposium. 28.
https://digitalscholar.lsuhsc.edu/sommrd/2019/posters/28
Functional Characterization of Mul1, a Ubiquitin Ligase Involved in Mitochondrial Recycling
Medical Education Building, LSUHSC-NO
Mitochondria play a crucial role in multiple cellular processes. Aside from supporting aerobic respiration, mitochondria are involved in steroid synthesis, calcium homeostasis and induction of apoptosis. They are highly dynamic organelles that undergo fission, fusion and distribute throughout the cell using the cytoskeletal network. Defects in mitochondrial dynamics can result in accumulation of dysfunctional mitochondria and enhanced oxidative stress, ultimately leading to neurodegenerative diseases such as Charcot-Marie-Tooth disease and autosomal dominant optic atrophy. Mul1 (Mulan) is a mitochondrial E3 ubiquitin ligase that functions in mitochondrial dynamics and integrity. Mul1 is expressed in all tissues, with the highest abundance in the heart, where mitochondrial quality control is particularly acute. Ectopic expression of Mul1 in HeLa cells leads to defects in both mitochondrial morphology and subcellular distribution. Mul1 localizes to the outer mitochondrial membrane and contains two transmembrane domains, and a C terminus that faces the cytosol. Mul1 exerts its regulatory effects by ubiquitination, which is a three-step process involving ubiquitin activation through E1 activating enzyme, transfer of a high energy ubiquitin thioester to E2 carrier protein to form E2~ubiquitin thioester and conjugation of E2-bound ubiquitin to target protein. The last step is catalyzed by E3 ubiquitin ligases. Previous work suggests that Mul1 specifically mediates conjugation of Mfn2 (mitofusin2) with ubiquitin. However, a recent retraction by Lokireddy et al. questions Mul1 conjugation of Mfn2. The aim of this study was to enzymatically characterize the mechanism of Mul1 ubiquitin conjugation. The cytoplasmic domain of Mul1 (accession number NP_078820, residues 262-352) was optimized for bacterial expression and subcloned into a pGEX expression vector. Recombinant GST-Mul1 was expressed in Escherichia coli BL21 (DE3) cells after induction with IPTG. The resulting protein was purified by affinity chromatography using glutathione sepharose and the GST was subsequently cleaved using thrombin. Mul1 was then resolved from unbound GST by affinity chromatography and used in subsequent experiments. Additionally, cytoplasmic domains of Mfn1 (accession number NM_033540, residues 1-580) and Mfn2 (residues 1-599, accession number NM_014874) were subcloned into pGEX, expressed and purified in a similar manner. A 125I-polyubiquitin chain formation functional assay was used to identify Ubc5A as a cognate E2 ubiquitin carrier for Mul1. Activity assays were also used to test whether recombinant mitofusin proteins can serve as substrates for Mul1-dependent conjugation with ubiquitin. Results showed that Mul1 shows broad linkage specificity in the assembly of free polyubiquitin chains in the absence of substrate. The assembly of unanchored ubiquitin chains was inhibited by Mfn2 but not by Mfn1, which suggests that the former binds Mul1 but does not serve as a substrate for ubiquitin conjugation.
Comments
Mentor: Dr. Arthur L. Haas (Department of Biochemistry & Molecular Biology)