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Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.

Li W, Bengtson MH, Ulbrich A, Matsuda A, Reddy VA, Orth A, Chanda SK, Batalov S, Joazeiro CA - PLoS ONE (2008)

Bottom Line: We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane.Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub.These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

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Identification of MULAN as a novel regulator of mitochondrial dynamics.A) Left 2 panels: Mitochondrial perinuclear clustering (a consequence of defective trafficking) results from siRNA-mediated knockdown of KIF5B, but not of KIF11. HeLa cells were transfected with the indicated siRNAs and mitochondria were visualized with MitoTracker Red. Right 2 panels: A cell-based imaging screen of E3 cDNA and shDNA genome-wide collections led to the identification of MULAN, a RING finger protein whose ectopic expression resulted in mitochondrial perinuclear clustering and fragmentation. For the screen, HeLa cells were co-transfected with mitochondrial-targeted RFP (MT-RFP) and with the E3 collections. “Control E3” refers to a random E3 cDNA which did not affect mitochondrial dynamics in the screen. B) Mitochondrial perinuclear clustering in response to MULAN ectopic expression apparently does not result from disruption of the microtubule network. HeLa cells were co-transfected with MT-RFP and either vector control or MULAN wild type cDNA. Cells were immuno-stained with anti-α-tubulin antibody to visualize the microtubule network. Green, tubulin staining; red, mitochondria. C) MULAN's schematic domain structure. Amino acid numbers indicated below. TMD, transmembrane domain. RNF, RING finger. D) MULAN's RNF has in vitro E3 activity. Reactions utilized GST fusions with the MULAN or c-Cbl RNFs as E3s. Activity was dependent on an intact RNF, since it was not detected in reactions using the MULAN C339A RNF mutant. c-Cbl's RNF was used as a positive control. Negative controls were reactions lacking an E3 (No E3) or reactions added to SDS sample buffer at t = 0 (unreacted). E) MULAN's RNF is required for the regulation of mitochondrial dynamics. NIH3T3 cells were transfected with vector, wild type MULAN cDNA or RNF-mutant cDNAs, together with MT-RFP. Cells were fixed at 24 h post-transfection and the fraction of RFP-positive cells exhibiting perinuclear-clustered mitochondria was scored (% trafficking defect). Data represent the average of at least 350 cells per condition, from 3–5 random 20× fields. Results are representative of several experiments.
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pone-0001487-g001: Identification of MULAN as a novel regulator of mitochondrial dynamics.A) Left 2 panels: Mitochondrial perinuclear clustering (a consequence of defective trafficking) results from siRNA-mediated knockdown of KIF5B, but not of KIF11. HeLa cells were transfected with the indicated siRNAs and mitochondria were visualized with MitoTracker Red. Right 2 panels: A cell-based imaging screen of E3 cDNA and shDNA genome-wide collections led to the identification of MULAN, a RING finger protein whose ectopic expression resulted in mitochondrial perinuclear clustering and fragmentation. For the screen, HeLa cells were co-transfected with mitochondrial-targeted RFP (MT-RFP) and with the E3 collections. “Control E3” refers to a random E3 cDNA which did not affect mitochondrial dynamics in the screen. B) Mitochondrial perinuclear clustering in response to MULAN ectopic expression apparently does not result from disruption of the microtubule network. HeLa cells were co-transfected with MT-RFP and either vector control or MULAN wild type cDNA. Cells were immuno-stained with anti-α-tubulin antibody to visualize the microtubule network. Green, tubulin staining; red, mitochondria. C) MULAN's schematic domain structure. Amino acid numbers indicated below. TMD, transmembrane domain. RNF, RING finger. D) MULAN's RNF has in vitro E3 activity. Reactions utilized GST fusions with the MULAN or c-Cbl RNFs as E3s. Activity was dependent on an intact RNF, since it was not detected in reactions using the MULAN C339A RNF mutant. c-Cbl's RNF was used as a positive control. Negative controls were reactions lacking an E3 (No E3) or reactions added to SDS sample buffer at t = 0 (unreacted). E) MULAN's RNF is required for the regulation of mitochondrial dynamics. NIH3T3 cells were transfected with vector, wild type MULAN cDNA or RNF-mutant cDNAs, together with MT-RFP. Cells were fixed at 24 h post-transfection and the fraction of RFP-positive cells exhibiting perinuclear-clustered mitochondria was scored (% trafficking defect). Data represent the average of at least 350 cells per condition, from 3–5 random 20× fields. Results are representative of several experiments.

Mentions: In one such screen, we set out to identify novel E3s that regulate mitochondrial dynamics. Mitochondria divide, fuse and move around cells [18]–[20], processes whose relevance is underscored by the observation that mutations in the cognate proteins can lead to disease, in particular neuronal dysfunction [19]. Mitochondria utilize the cytoskeleton as tracks for directional movement –in mammalian cells, they are distributed mainly along microtubules, with kinesins (KIF) 1B and 5B having been identified as the anterograde (plus end) trafficking motors [21]. A phenotype that is characteristic of defective mitochondrial anterograde trafficking is the perinuclear clustering of the organelle, which we observed following transfection of HeLa cells with siRNA against KIF5B (a gift of Dr. Jiang Wei, Burnham Institute; [22]); in contrast, mitochondrial trafficking was not impaired in cells transfected with siRNA to KIF11, which has not been implicated in mitochondrial trafficking (Fig. 1A).


Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.

Li W, Bengtson MH, Ulbrich A, Matsuda A, Reddy VA, Orth A, Chanda SK, Batalov S, Joazeiro CA - PLoS ONE (2008)

Identification of MULAN as a novel regulator of mitochondrial dynamics.A) Left 2 panels: Mitochondrial perinuclear clustering (a consequence of defective trafficking) results from siRNA-mediated knockdown of KIF5B, but not of KIF11. HeLa cells were transfected with the indicated siRNAs and mitochondria were visualized with MitoTracker Red. Right 2 panels: A cell-based imaging screen of E3 cDNA and shDNA genome-wide collections led to the identification of MULAN, a RING finger protein whose ectopic expression resulted in mitochondrial perinuclear clustering and fragmentation. For the screen, HeLa cells were co-transfected with mitochondrial-targeted RFP (MT-RFP) and with the E3 collections. “Control E3” refers to a random E3 cDNA which did not affect mitochondrial dynamics in the screen. B) Mitochondrial perinuclear clustering in response to MULAN ectopic expression apparently does not result from disruption of the microtubule network. HeLa cells were co-transfected with MT-RFP and either vector control or MULAN wild type cDNA. Cells were immuno-stained with anti-α-tubulin antibody to visualize the microtubule network. Green, tubulin staining; red, mitochondria. C) MULAN's schematic domain structure. Amino acid numbers indicated below. TMD, transmembrane domain. RNF, RING finger. D) MULAN's RNF has in vitro E3 activity. Reactions utilized GST fusions with the MULAN or c-Cbl RNFs as E3s. Activity was dependent on an intact RNF, since it was not detected in reactions using the MULAN C339A RNF mutant. c-Cbl's RNF was used as a positive control. Negative controls were reactions lacking an E3 (No E3) or reactions added to SDS sample buffer at t = 0 (unreacted). E) MULAN's RNF is required for the regulation of mitochondrial dynamics. NIH3T3 cells were transfected with vector, wild type MULAN cDNA or RNF-mutant cDNAs, together with MT-RFP. Cells were fixed at 24 h post-transfection and the fraction of RFP-positive cells exhibiting perinuclear-clustered mitochondria was scored (% trafficking defect). Data represent the average of at least 350 cells per condition, from 3–5 random 20× fields. Results are representative of several experiments.
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Related In: Results  -  Collection

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pone-0001487-g001: Identification of MULAN as a novel regulator of mitochondrial dynamics.A) Left 2 panels: Mitochondrial perinuclear clustering (a consequence of defective trafficking) results from siRNA-mediated knockdown of KIF5B, but not of KIF11. HeLa cells were transfected with the indicated siRNAs and mitochondria were visualized with MitoTracker Red. Right 2 panels: A cell-based imaging screen of E3 cDNA and shDNA genome-wide collections led to the identification of MULAN, a RING finger protein whose ectopic expression resulted in mitochondrial perinuclear clustering and fragmentation. For the screen, HeLa cells were co-transfected with mitochondrial-targeted RFP (MT-RFP) and with the E3 collections. “Control E3” refers to a random E3 cDNA which did not affect mitochondrial dynamics in the screen. B) Mitochondrial perinuclear clustering in response to MULAN ectopic expression apparently does not result from disruption of the microtubule network. HeLa cells were co-transfected with MT-RFP and either vector control or MULAN wild type cDNA. Cells were immuno-stained with anti-α-tubulin antibody to visualize the microtubule network. Green, tubulin staining; red, mitochondria. C) MULAN's schematic domain structure. Amino acid numbers indicated below. TMD, transmembrane domain. RNF, RING finger. D) MULAN's RNF has in vitro E3 activity. Reactions utilized GST fusions with the MULAN or c-Cbl RNFs as E3s. Activity was dependent on an intact RNF, since it was not detected in reactions using the MULAN C339A RNF mutant. c-Cbl's RNF was used as a positive control. Negative controls were reactions lacking an E3 (No E3) or reactions added to SDS sample buffer at t = 0 (unreacted). E) MULAN's RNF is required for the regulation of mitochondrial dynamics. NIH3T3 cells were transfected with vector, wild type MULAN cDNA or RNF-mutant cDNAs, together with MT-RFP. Cells were fixed at 24 h post-transfection and the fraction of RFP-positive cells exhibiting perinuclear-clustered mitochondria was scored (% trafficking defect). Data represent the average of at least 350 cells per condition, from 3–5 random 20× fields. Results are representative of several experiments.
Mentions: In one such screen, we set out to identify novel E3s that regulate mitochondrial dynamics. Mitochondria divide, fuse and move around cells [18]–[20], processes whose relevance is underscored by the observation that mutations in the cognate proteins can lead to disease, in particular neuronal dysfunction [19]. Mitochondria utilize the cytoskeleton as tracks for directional movement –in mammalian cells, they are distributed mainly along microtubules, with kinesins (KIF) 1B and 5B having been identified as the anterograde (plus end) trafficking motors [21]. A phenotype that is characteristic of defective mitochondrial anterograde trafficking is the perinuclear clustering of the organelle, which we observed following transfection of HeLa cells with siRNA against KIF5B (a gift of Dr. Jiang Wei, Burnham Institute; [22]); in contrast, mitochondrial trafficking was not impaired in cells transfected with siRNA to KIF11, which has not been implicated in mitochondrial trafficking (Fig. 1A).

Bottom Line: We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane.Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub.These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

Show MeSH
Related in: MedlinePlus