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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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MULAN is a mitochondrial outer membrane (MOM) protein with a cytosolic-facing C-terminal RING-finger.A) MULAN colocalizes with MT-RFP/MT-GFP. NIH3T3 cells were transfected with Flag-tagged MULAN or untagged MULAN 1-301, together with MT-RFP (top panels) or MT-GFP (bottom panels), followed by immunostaining with antibody against Flag (top; green) or MULAN (bottom; red). B) Left panel: MULAN-Flag co-sediments with the mitochondrial protein, Tom20, in sucrose gradient. 293 cells transfected with MULAN-Flag were subfractionated by sucrose gradient. MULAN-Flag, the MOM protein Tom20, and Golgin 97 in each fraction were detected by western blot. Right panel: endogenous MULAN co-fractionates with mitochondria in sucrose gradient. 293F cells were Dounce-homogenized and centrifuged at 750×g to pellet nuclei and unbroken cells. The post-nuclear supernatant was centrifuged at 12,500×g to obtain the heavy membrane (HM) and the cytosolic/light membrane (LM) fractions. The HM fraction was subjected to sucrose gradient to further purify mitochondria. Equal protein amounts (40 µg) were fractionated by SDS-PAGE and blotted with anti-MULAN antibody to detect endogenous MULAN. Cytochrome c, Golgin 97, EEA1 and tubulin were used as markers for mitochondria, Golgi, endosomes and cytosol, respectively. Calreticulin and Sec61 were both used as ER markers. C) Immuno-electron microscopy of MULAN. COS7 cells transfected or not with MULAN-Flag were fixed and stained with anti-Flag antibody followed by gold-conjugated secondary antibody for immuno-EM analysis. Left panel: BSA only (no primary antibody) control; inset: mitochondrion from an untransfected cell. Right panel: mitochondria expressing MULAN-Flag. Scale bars are indicated. D) MULAN's predicted transmembrane domains (TMDs) mediate localization to mitochondria. NIH3T3 cells were co-transfected with Flag-tagged MULAN deletion mutants (green) and MT-RFP (red). Deletion of TMD1 (MULAN Δ2-29; top row) or of TMD2 (MULAN Δ242-259; middle row) led to partial mislocalization of MULAN. The combined deletion of both TMDs led to complete mislocalization of MULAN to the cytosol (MULAN Δ2-29+Δ242-259; bottom row). E) MULAN is a mitochondrial outer membrane (MOM) protein. Mitochondria from 293 cells transfected with MULAN-Flag were purified by sucrose gradient. Intact mitochondria were treated with trypsin for the indicated time, in the presence or absence of Triton X-100. The MULAN C-terminus was readily susceptible to trypsin digestion in intact mitochondria, indicating that it sits in the MOM facing the cytosol. Controls are the MOM protein, Tom20, and the intermembrane space protein, Smac. Smac only becomes sensitive to trypsin upon lysis of mitochondria with 0.5% Triton X-100 (lower panel). F) Topological model for MULAN on the MOM, indicating its transmembrane domains (red) and RNF (green). The cytosolic-exposed RNF can have access to the remaining components of the Ub system.
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pone-0001487-g003: MULAN is a mitochondrial outer membrane (MOM) protein with a cytosolic-facing C-terminal RING-finger.A) MULAN colocalizes with MT-RFP/MT-GFP. NIH3T3 cells were transfected with Flag-tagged MULAN or untagged MULAN 1-301, together with MT-RFP (top panels) or MT-GFP (bottom panels), followed by immunostaining with antibody against Flag (top; green) or MULAN (bottom; red). B) Left panel: MULAN-Flag co-sediments with the mitochondrial protein, Tom20, in sucrose gradient. 293 cells transfected with MULAN-Flag were subfractionated by sucrose gradient. MULAN-Flag, the MOM protein Tom20, and Golgin 97 in each fraction were detected by western blot. Right panel: endogenous MULAN co-fractionates with mitochondria in sucrose gradient. 293F cells were Dounce-homogenized and centrifuged at 750×g to pellet nuclei and unbroken cells. The post-nuclear supernatant was centrifuged at 12,500×g to obtain the heavy membrane (HM) and the cytosolic/light membrane (LM) fractions. The HM fraction was subjected to sucrose gradient to further purify mitochondria. Equal protein amounts (40 µg) were fractionated by SDS-PAGE and blotted with anti-MULAN antibody to detect endogenous MULAN. Cytochrome c, Golgin 97, EEA1 and tubulin were used as markers for mitochondria, Golgi, endosomes and cytosol, respectively. Calreticulin and Sec61 were both used as ER markers. C) Immuno-electron microscopy of MULAN. COS7 cells transfected or not with MULAN-Flag were fixed and stained with anti-Flag antibody followed by gold-conjugated secondary antibody for immuno-EM analysis. Left panel: BSA only (no primary antibody) control; inset: mitochondrion from an untransfected cell. Right panel: mitochondria expressing MULAN-Flag. Scale bars are indicated. D) MULAN's predicted transmembrane domains (TMDs) mediate localization to mitochondria. NIH3T3 cells were co-transfected with Flag-tagged MULAN deletion mutants (green) and MT-RFP (red). Deletion of TMD1 (MULAN Δ2-29; top row) or of TMD2 (MULAN Δ242-259; middle row) led to partial mislocalization of MULAN. The combined deletion of both TMDs led to complete mislocalization of MULAN to the cytosol (MULAN Δ2-29+Δ242-259; bottom row). E) MULAN is a mitochondrial outer membrane (MOM) protein. Mitochondria from 293 cells transfected with MULAN-Flag were purified by sucrose gradient. Intact mitochondria were treated with trypsin for the indicated time, in the presence or absence of Triton X-100. The MULAN C-terminus was readily susceptible to trypsin digestion in intact mitochondria, indicating that it sits in the MOM facing the cytosol. Controls are the MOM protein, Tom20, and the intermembrane space protein, Smac. Smac only becomes sensitive to trypsin upon lysis of mitochondria with 0.5% Triton X-100 (lower panel). F) Topological model for MULAN on the MOM, indicating its transmembrane domains (red) and RNF (green). The cytosolic-exposed RNF can have access to the remaining components of the Ub system.

Mentions: To begin elucidating the mechanism by which MULAN affects mitochondrial dynamics, we determined its subcellular localization. Earlier results had already revealed that the protein's distribution was reminiscent of mitochondrial staining patterns. Several lines of evidence indeed indicate that MULAN is a mitochondrial protein: (i) the signal of C-terminal Flag-tagged MULAN completely overlapped with that of co-transfected MT-RFP (Fig. 3A, upper panels). The untagged MULAN 1-301 protein, whose levels were high enough to be detected by immunocytochemistry using anti-MULAN antibody, also colocalized with MT-GFP (Fig. 3A, lower panels). Thus, both an untagged and the C-terminal tagged E3 co-localized with mitochondria. The specificity of MULAN localization is also revealed by the observation that neither protein colocalized with the Golgi marker lectin-Alexa 488 (Fig. S1E); (ii) lysates of 293 cells overexpressing MULAN-Flag were subjected to centrifugation in sucrose gradient. Gradient fractions were run in SDS-PAGE and analyzed by western-blot with different antibodies, revealing that MULAN's sedimentation was similar to that of the mitochondrial protein Tom20 but not of the Golgi marker, Golgin 97 (Fig. 3B, left panel); (iii) detergent-free lysates of untransfected 293F cells were first fractionated by differential centrifugation. Mitochondria in the heavy membrane (HM) fraction were further purified by sucrose gradient centrifugation. Samples of the whole cell extract, “mitochondrial” fraction, and cytosolic and light membrane (LM) fraction were normalized for protein amount and analyzed by western-blot using antibodies against cytochrome c, Golgin 97, calreticulin/Sec61, tubulin or EEA1 (markers for mitochondria, Golgi, ER, cytosol and endosomes, respectively; Fig. 3B, right panel). Consistently with the previous observations, the results show that endogenous MULAN was abundant in the fraction enriched for mitochondria, but was not detected in the fraction enriched for Golgi, ER, cytosol and endosomes. Although some Golgi material contaminated the “mitochondrial” fraction, immunocytochemistry analyses also revealed that MULAN-Flag did not detectably colocalize with lectin in intact cells (Fig. S1E). In the case of the ER, there was also contamination of the mitochondrial fraction with soluble ER luminal material (calreticulin) and the ER signal in immunocytochemistry was too disperse to allow complete resolution from mitochondria (Fig. S1F). However, we do not expect a significant fraction of MULAN to be localized to the ER, since this organelle's membrane-resident marker Sec61 was not detected in the purified mitochondrial fraction enriched for MULAN, and MULAN was not detected in the LM fraction enriched for the ER (Fig. 1B); (vi) lastly, immuno-electron microscopy analyses demonstrated that the MULAN signal concentrated specifically around the circumference of mitochondria (Figs. 3C and S1). The finding that MULAN localizes to mitochondria is interesting because only one other transmembrane E3 has been shown to reside in this organelle [9], [11]. These results suggest that MULAN's effects on mitochondrial dynamics are direct.


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)

MULAN is a mitochondrial outer membrane (MOM) protein with a cytosolic-facing C-terminal RING-finger.A) MULAN colocalizes with MT-RFP/MT-GFP. NIH3T3 cells were transfected with Flag-tagged MULAN or untagged MULAN 1-301, together with MT-RFP (top panels) or MT-GFP (bottom panels), followed by immunostaining with antibody against Flag (top; green) or MULAN (bottom; red). B) Left panel: MULAN-Flag co-sediments with the mitochondrial protein, Tom20, in sucrose gradient. 293 cells transfected with MULAN-Flag were subfractionated by sucrose gradient. MULAN-Flag, the MOM protein Tom20, and Golgin 97 in each fraction were detected by western blot. Right panel: endogenous MULAN co-fractionates with mitochondria in sucrose gradient. 293F cells were Dounce-homogenized and centrifuged at 750×g to pellet nuclei and unbroken cells. The post-nuclear supernatant was centrifuged at 12,500×g to obtain the heavy membrane (HM) and the cytosolic/light membrane (LM) fractions. The HM fraction was subjected to sucrose gradient to further purify mitochondria. Equal protein amounts (40 µg) were fractionated by SDS-PAGE and blotted with anti-MULAN antibody to detect endogenous MULAN. Cytochrome c, Golgin 97, EEA1 and tubulin were used as markers for mitochondria, Golgi, endosomes and cytosol, respectively. Calreticulin and Sec61 were both used as ER markers. C) Immuno-electron microscopy of MULAN. COS7 cells transfected or not with MULAN-Flag were fixed and stained with anti-Flag antibody followed by gold-conjugated secondary antibody for immuno-EM analysis. Left panel: BSA only (no primary antibody) control; inset: mitochondrion from an untransfected cell. Right panel: mitochondria expressing MULAN-Flag. Scale bars are indicated. D) MULAN's predicted transmembrane domains (TMDs) mediate localization to mitochondria. NIH3T3 cells were co-transfected with Flag-tagged MULAN deletion mutants (green) and MT-RFP (red). Deletion of TMD1 (MULAN Δ2-29; top row) or of TMD2 (MULAN Δ242-259; middle row) led to partial mislocalization of MULAN. The combined deletion of both TMDs led to complete mislocalization of MULAN to the cytosol (MULAN Δ2-29+Δ242-259; bottom row). E) MULAN is a mitochondrial outer membrane (MOM) protein. Mitochondria from 293 cells transfected with MULAN-Flag were purified by sucrose gradient. Intact mitochondria were treated with trypsin for the indicated time, in the presence or absence of Triton X-100. The MULAN C-terminus was readily susceptible to trypsin digestion in intact mitochondria, indicating that it sits in the MOM facing the cytosol. Controls are the MOM protein, Tom20, and the intermembrane space protein, Smac. Smac only becomes sensitive to trypsin upon lysis of mitochondria with 0.5% Triton X-100 (lower panel). F) Topological model for MULAN on the MOM, indicating its transmembrane domains (red) and RNF (green). The cytosolic-exposed RNF can have access to the remaining components of the Ub system.
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pone-0001487-g003: MULAN is a mitochondrial outer membrane (MOM) protein with a cytosolic-facing C-terminal RING-finger.A) MULAN colocalizes with MT-RFP/MT-GFP. NIH3T3 cells were transfected with Flag-tagged MULAN or untagged MULAN 1-301, together with MT-RFP (top panels) or MT-GFP (bottom panels), followed by immunostaining with antibody against Flag (top; green) or MULAN (bottom; red). B) Left panel: MULAN-Flag co-sediments with the mitochondrial protein, Tom20, in sucrose gradient. 293 cells transfected with MULAN-Flag were subfractionated by sucrose gradient. MULAN-Flag, the MOM protein Tom20, and Golgin 97 in each fraction were detected by western blot. Right panel: endogenous MULAN co-fractionates with mitochondria in sucrose gradient. 293F cells were Dounce-homogenized and centrifuged at 750×g to pellet nuclei and unbroken cells. The post-nuclear supernatant was centrifuged at 12,500×g to obtain the heavy membrane (HM) and the cytosolic/light membrane (LM) fractions. The HM fraction was subjected to sucrose gradient to further purify mitochondria. Equal protein amounts (40 µg) were fractionated by SDS-PAGE and blotted with anti-MULAN antibody to detect endogenous MULAN. Cytochrome c, Golgin 97, EEA1 and tubulin were used as markers for mitochondria, Golgi, endosomes and cytosol, respectively. Calreticulin and Sec61 were both used as ER markers. C) Immuno-electron microscopy of MULAN. COS7 cells transfected or not with MULAN-Flag were fixed and stained with anti-Flag antibody followed by gold-conjugated secondary antibody for immuno-EM analysis. Left panel: BSA only (no primary antibody) control; inset: mitochondrion from an untransfected cell. Right panel: mitochondria expressing MULAN-Flag. Scale bars are indicated. D) MULAN's predicted transmembrane domains (TMDs) mediate localization to mitochondria. NIH3T3 cells were co-transfected with Flag-tagged MULAN deletion mutants (green) and MT-RFP (red). Deletion of TMD1 (MULAN Δ2-29; top row) or of TMD2 (MULAN Δ242-259; middle row) led to partial mislocalization of MULAN. The combined deletion of both TMDs led to complete mislocalization of MULAN to the cytosol (MULAN Δ2-29+Δ242-259; bottom row). E) MULAN is a mitochondrial outer membrane (MOM) protein. Mitochondria from 293 cells transfected with MULAN-Flag were purified by sucrose gradient. Intact mitochondria were treated with trypsin for the indicated time, in the presence or absence of Triton X-100. The MULAN C-terminus was readily susceptible to trypsin digestion in intact mitochondria, indicating that it sits in the MOM facing the cytosol. Controls are the MOM protein, Tom20, and the intermembrane space protein, Smac. Smac only becomes sensitive to trypsin upon lysis of mitochondria with 0.5% Triton X-100 (lower panel). F) Topological model for MULAN on the MOM, indicating its transmembrane domains (red) and RNF (green). The cytosolic-exposed RNF can have access to the remaining components of the Ub system.
Mentions: To begin elucidating the mechanism by which MULAN affects mitochondrial dynamics, we determined its subcellular localization. Earlier results had already revealed that the protein's distribution was reminiscent of mitochondrial staining patterns. Several lines of evidence indeed indicate that MULAN is a mitochondrial protein: (i) the signal of C-terminal Flag-tagged MULAN completely overlapped with that of co-transfected MT-RFP (Fig. 3A, upper panels). The untagged MULAN 1-301 protein, whose levels were high enough to be detected by immunocytochemistry using anti-MULAN antibody, also colocalized with MT-GFP (Fig. 3A, lower panels). Thus, both an untagged and the C-terminal tagged E3 co-localized with mitochondria. The specificity of MULAN localization is also revealed by the observation that neither protein colocalized with the Golgi marker lectin-Alexa 488 (Fig. S1E); (ii) lysates of 293 cells overexpressing MULAN-Flag were subjected to centrifugation in sucrose gradient. Gradient fractions were run in SDS-PAGE and analyzed by western-blot with different antibodies, revealing that MULAN's sedimentation was similar to that of the mitochondrial protein Tom20 but not of the Golgi marker, Golgin 97 (Fig. 3B, left panel); (iii) detergent-free lysates of untransfected 293F cells were first fractionated by differential centrifugation. Mitochondria in the heavy membrane (HM) fraction were further purified by sucrose gradient centrifugation. Samples of the whole cell extract, “mitochondrial” fraction, and cytosolic and light membrane (LM) fraction were normalized for protein amount and analyzed by western-blot using antibodies against cytochrome c, Golgin 97, calreticulin/Sec61, tubulin or EEA1 (markers for mitochondria, Golgi, ER, cytosol and endosomes, respectively; Fig. 3B, right panel). Consistently with the previous observations, the results show that endogenous MULAN was abundant in the fraction enriched for mitochondria, but was not detected in the fraction enriched for Golgi, ER, cytosol and endosomes. Although some Golgi material contaminated the “mitochondrial” fraction, immunocytochemistry analyses also revealed that MULAN-Flag did not detectably colocalize with lectin in intact cells (Fig. S1E). In the case of the ER, there was also contamination of the mitochondrial fraction with soluble ER luminal material (calreticulin) and the ER signal in immunocytochemistry was too disperse to allow complete resolution from mitochondria (Fig. S1F). However, we do not expect a significant fraction of MULAN to be localized to the ER, since this organelle's membrane-resident marker Sec61 was not detected in the purified mitochondrial fraction enriched for MULAN, and MULAN was not detected in the LM fraction enriched for the ER (Fig. 1B); (vi) lastly, immuno-electron microscopy analyses demonstrated that the MULAN signal concentrated specifically around the circumference of mitochondria (Figs. 3C and S1). The finding that MULAN localizes to mitochondria is interesting because only one other transmembrane E3 has been shown to reside in this organelle [9], [11]. These results suggest that MULAN's effects on mitochondrial dynamics are direct.

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