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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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Knockdown of endogenous MULAN perturbs mitochondrial dynamics.A) Blue bars: MULAN mRNA knockdown. RNA was extracted from HeLa cells 48 h after transfection with siRNA oligos targeting two different MULAN sequences, or with a negative control siRNA (siScrambled). Knockdown efficiency was determined by quantitative real-time RT-PCR. Results were normalized to the level of the 36B4 mRNA, which was amplified in the same multiplex reaction, as an internal control. Purple bars: MULAN knockdown leads to mitochondrial perinuclear clustering. HeLa cells were transfected with the indicated siRNAs on day 1, followed by MT-RFP on day 2. Cells were fixed on day 3 and the percentage of RFP-positive cells with perinuclear-clustered mitochondria was scored. B) The microtubule network is apparently intact in MULAN-knockdown cells. As in “A”, except that cells were fixed and stained with anti-α-tubulin antibody for analysis (green). C) Both MULAN ectopic expression and endogenous knockdown indicate its role in mitochondrial dynamics. For ectopic expression, HeLa cells were transfected with vector or MULAN wild type cDNA together with MT-RFP. Cells were fixed for analysis 24 h post-transfection. For siRNA-mediated knockdown, cells were transfected with siScrambled or siMULAN1 on day 1 and with MT-RFP on day 2. Cells were fixed on day 3. Mitochondrial fragmentation and clustering were quantitated under 100× field. At least 100 cells were counted per condition and data represent average of three independent transfections.
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pone-0001487-g002: Knockdown of endogenous MULAN perturbs mitochondrial dynamics.A) Blue bars: MULAN mRNA knockdown. RNA was extracted from HeLa cells 48 h after transfection with siRNA oligos targeting two different MULAN sequences, or with a negative control siRNA (siScrambled). Knockdown efficiency was determined by quantitative real-time RT-PCR. Results were normalized to the level of the 36B4 mRNA, which was amplified in the same multiplex reaction, as an internal control. Purple bars: MULAN knockdown leads to mitochondrial perinuclear clustering. HeLa cells were transfected with the indicated siRNAs on day 1, followed by MT-RFP on day 2. Cells were fixed on day 3 and the percentage of RFP-positive cells with perinuclear-clustered mitochondria was scored. B) The microtubule network is apparently intact in MULAN-knockdown cells. As in “A”, except that cells were fixed and stained with anti-α-tubulin antibody for analysis (green). C) Both MULAN ectopic expression and endogenous knockdown indicate its role in mitochondrial dynamics. For ectopic expression, HeLa cells were transfected with vector or MULAN wild type cDNA together with MT-RFP. Cells were fixed for analysis 24 h post-transfection. For siRNA-mediated knockdown, cells were transfected with siScrambled or siMULAN1 on day 1 and with MT-RFP on day 2. Cells were fixed on day 3. Mitochondrial fragmentation and clustering were quantitated under 100× field. At least 100 cells were counted per condition and data represent average of three independent transfections.

Mentions: To confirm that endogenous MULAN plays a role in mitochondrial dynamics, we generated two different siRNA, siMULAN1 and siMULAN2, which led to greater than 70% reduction of the endogenous mRNA level two days after transfection of HeLa cells (Fig. 2A; knockdown was also observed at the protein level, Fig. S2C). The phenotype of MULAN knocked-down cells exhibited similarities to that of cells ectopically expressing the E3, which is not unprecedented (e.g., [23], [24]) and could be due to a dominant negative effect of the ectopically expressed protein (Fig. 2 and Fig. S3). Mitochondria in 80–90% of MULAN siRNA-transfected cells were clustered around the nucleus, although clustering was less compact compared to that seen in response to ectopic MULAN expression; in contrast, only 5–12% of cells transfected with the control non-silencing siRNA exhibited this phenotype (siScrambled; Figs. 2A and 2C). Immunostaining with anti-α-tubulin antibody in cells knocked down for MULAN did not reveal an obvious disruption of the microtubule network (Fig. 2B). The fact that the knockdown phenotype was specific to siRNA against MULAN and that it was observed with two different siRNA sequences argues against an off-target effect and confirms a role for MULAN in the regulation of mitochondrial dynamics.


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)

Knockdown of endogenous MULAN perturbs mitochondrial dynamics.A) Blue bars: MULAN mRNA knockdown. RNA was extracted from HeLa cells 48 h after transfection with siRNA oligos targeting two different MULAN sequences, or with a negative control siRNA (siScrambled). Knockdown efficiency was determined by quantitative real-time RT-PCR. Results were normalized to the level of the 36B4 mRNA, which was amplified in the same multiplex reaction, as an internal control. Purple bars: MULAN knockdown leads to mitochondrial perinuclear clustering. HeLa cells were transfected with the indicated siRNAs on day 1, followed by MT-RFP on day 2. Cells were fixed on day 3 and the percentage of RFP-positive cells with perinuclear-clustered mitochondria was scored. B) The microtubule network is apparently intact in MULAN-knockdown cells. As in “A”, except that cells were fixed and stained with anti-α-tubulin antibody for analysis (green). C) Both MULAN ectopic expression and endogenous knockdown indicate its role in mitochondrial dynamics. For ectopic expression, HeLa cells were transfected with vector or MULAN wild type cDNA together with MT-RFP. Cells were fixed for analysis 24 h post-transfection. For siRNA-mediated knockdown, cells were transfected with siScrambled or siMULAN1 on day 1 and with MT-RFP on day 2. Cells were fixed on day 3. Mitochondrial fragmentation and clustering were quantitated under 100× field. At least 100 cells were counted per condition and data represent average of three independent transfections.
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Related In: Results  -  Collection

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pone-0001487-g002: Knockdown of endogenous MULAN perturbs mitochondrial dynamics.A) Blue bars: MULAN mRNA knockdown. RNA was extracted from HeLa cells 48 h after transfection with siRNA oligos targeting two different MULAN sequences, or with a negative control siRNA (siScrambled). Knockdown efficiency was determined by quantitative real-time RT-PCR. Results were normalized to the level of the 36B4 mRNA, which was amplified in the same multiplex reaction, as an internal control. Purple bars: MULAN knockdown leads to mitochondrial perinuclear clustering. HeLa cells were transfected with the indicated siRNAs on day 1, followed by MT-RFP on day 2. Cells were fixed on day 3 and the percentage of RFP-positive cells with perinuclear-clustered mitochondria was scored. B) The microtubule network is apparently intact in MULAN-knockdown cells. As in “A”, except that cells were fixed and stained with anti-α-tubulin antibody for analysis (green). C) Both MULAN ectopic expression and endogenous knockdown indicate its role in mitochondrial dynamics. For ectopic expression, HeLa cells were transfected with vector or MULAN wild type cDNA together with MT-RFP. Cells were fixed for analysis 24 h post-transfection. For siRNA-mediated knockdown, cells were transfected with siScrambled or siMULAN1 on day 1 and with MT-RFP on day 2. Cells were fixed on day 3. Mitochondrial fragmentation and clustering were quantitated under 100× field. At least 100 cells were counted per condition and data represent average of three independent transfections.
Mentions: To confirm that endogenous MULAN plays a role in mitochondrial dynamics, we generated two different siRNA, siMULAN1 and siMULAN2, which led to greater than 70% reduction of the endogenous mRNA level two days after transfection of HeLa cells (Fig. 2A; knockdown was also observed at the protein level, Fig. S2C). The phenotype of MULAN knocked-down cells exhibited similarities to that of cells ectopically expressing the E3, which is not unprecedented (e.g., [23], [24]) and could be due to a dominant negative effect of the ectopically expressed protein (Fig. 2 and Fig. S3). Mitochondria in 80–90% of MULAN siRNA-transfected cells were clustered around the nucleus, although clustering was less compact compared to that seen in response to ectopic MULAN expression; in contrast, only 5–12% of cells transfected with the control non-silencing siRNA exhibited this phenotype (siScrambled; Figs. 2A and 2C). Immunostaining with anti-α-tubulin antibody in cells knocked down for MULAN did not reveal an obvious disruption of the microtubule network (Fig. 2B). The fact that the knockdown phenotype was specific to siRNA against MULAN and that it was observed with two different siRNA sequences argues against an off-target effect and confirms a role for MULAN in the regulation of mitochondrial dynamics.

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