MITOL ubiquitinates mSOD1 in mitochondria. (A) Ubiquitinations of G93A and G85R by MITOL WT but not MITOL CS mutant. Lysates of COS-7 (left) or Neuro2a cells (right) transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibody. Whole lysates were immunoblotted with anti-myc antibody. (B) Ubiquitination of mSOD1 by MITOL mainly occurs in mitochondria. Cytosolic and mitochondrial fractions were isolated from COS-7 cells transfected with indicated vectors, each fraction was immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibody. Mitochondrial fractions and cytosolic fractions were immunoblotted with anti-myc or anti-Tim23 antibody (left). Neuro2a cells were transfected with mitochondria targeted-mSOD1 or ER-targeted mSOD1 with MITOL WT or control vector. Lysates were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibody (right). (C) MITOL directly ubiquitinates mSOD1 in vitro. In vitro ubiquitination assay was performed as described in Materials and Methods. Immunopurified MITOL-HA and mSOD1-FLAG were prepared from COS-7 cells transfected with MITOL- HA and mSOD1-FLAG, independently. Identification of E2s for MITOL-dependent ubiquitination of mSOD1 (left). MITOL WT-dependent ubiquitination of mSOD1 (right). (D) MULAN fails to ubiquitinate mSOD1. Mitochondrial fractions of Neuro2a cells transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, and immunoprecipitates were immunoblotted with anti-HA antibody. Inputs were immunoblotted with anti-myc or anti-V5 antibody.

MITOL knockdown induces mSOD1 accumulation and enhances mSOD1-dependent cell toxicity. (A) Accumulation of mSOD1 in mitochondria by siRNA-mediated MITOL knockdown. Neuro2a cells were cotransfected with mSOD1 and siRNAs (scramble or two different MITOL siRNAs). Mitochondrial fractions isolated from these cells were immunoblotted with anti-myc, anti-MITOL or anti-Tim23 antibody. The relative protein level of SOD1 shown in A was quantified by densitometry. Error bars, SD (n = 3). * p < 0.05; Student's t test. (B) Inhibition of mSOD1 ubiquitination by MITOL knockdown. Mitochondrial fractions of Neuro2a cells transfected with G93A-FLAG and siRNAs (scramble or MITOL siRNA) were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates and inputs were immunoblotted with indicated antibodies. (C) Inhibition of mSOD1 ubiquitination in MITOL-knockdown Neuro2a cells. Neuro2a cells stably expressing shMITOL or shGFP were transfected with indicated vectors. Human MITOL WT and CS were used as resistant vectors for mouse MITOL shRNA. Mitochondria fractions were immunoprecipitated with anti-FLAG antibody and immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibody. Inputs were immunoblotted with anti-MITOL antibody. (D) Delayed degradation of mSOD1 by MITOL depletion. Neuro2a cells were cotransfected with mSOD1 and siRNA (scramble or MITOL siRNA). Forty-eight hours after transfection, cells were treated with CHX (10 μg/ml) for indicated times, and lysates were analyzed by immunoblotting with anti-FLAG, anti-MITOL, or anti-tubulin antibody. The relative protein levels of mSOD1 were quantified by densitometry. Error bars, SD (n = 3). (E) Effects of MITOL depletion on ATP production and cell viability. Neuro2a cells were cotransfected with mSOD1 (G93A or G85R) and siRNA (scramble or MITOL siRNA). Twenty-four hours after transfection, cells were incubated in differentiation medium for 24 h. MTT assay and ATP production assay were performed. Error bars, SD (n = 3). * p < 0.05; Student's t test. (F) MITOL depletion enhances mSOD1-dependent ROS production. Twenty-four hours after cotransfection of Neuro2a cells with mSOD1 (G93A or G85R) or/and siRNAs (sc, scramble; siRNA, MITOL-specific siRNA) plus pDsRed-Mito, cells were incubated in differentiation medium for 24 h. Intracellular ROS generation in DsRed-Mito–positive Neuro2a cells was measured by flowcytometric analysis using H₂DCFDA staining. These represented results were from one of three experiments that produced similar results.

MITOL interacts with mSOD1 but not WT SOD1. (A) Partial colocalization of mSOD1 (G93A) with mitochondria. COS-7 cells coexpressing WT SOD1-FLAG or mSOD1-FLAG with a mitochondrial marker, pDsRed-Mito (top) or MITOL-HA (bottom), were immunostained with indicated antibodies. Right panels, insets show a magnification of the merge. Bars, 10 μm. (B) Accumulation of mSOD1 in mitochondria. Whole (W), cytosolic (C), and mitochondrial (M) fractions isolated from COS-7 cells expressing WT SOD1-FLAG or mSOD1-FLAG were immunoblotted with anti-FLAG, anti-tubulin, or anti-Tim23 mitochondrial marker antibodies. The relative protein levels of SOD1 in mitochondria and cytosolic fractions were quantified by densitometry (right panels). Errors bar, SD (n = 3). * p < 0.05; Student's t test. (C) Association of MITOL with mSOD1. Mitochondrial fractions isolated from COS-7 cells transfected with indicated vectors were immunoprecipitated with anti-myc antibody, and immunoprecipitates were immunoblotted with anti-FLAG or anti-myc antibody. Mitochondrial fractions (Input) were immunoblotted with anti-FLAG antibody to confirm the expression of SOD1-FLAG. (D) Association of endogenous MITOL with mSOD1. Neuro2a (1 × 10⁹) cells transfected with mSOD1-FLAG were treated with MG132 for 3 h. Mitochondrial fractions isolated from these cells were immunoprecipitated with control IgG or anti-MITOL antibody. Immunoprecipitates and mitochondrial fractions (Input) were immunoblotted with indicated antibodies.

MITOL overexpression promotes mSOD1 degradation. (A) MITOL overexpression decreases the amount of mSOD1 in mitochondria. Mitochondrial fractions isolated from Neuro2a cells transfected with indicated vectors were immunoblotted with anti-myc, anti-FLAG, or anti-Tim23 antibody. The relative protein level of mSOD1 with or without MITOL was quantified by densitometry (right). Error bars, SD (n = 3). * p <0.05; Student's t test. (B) Cycloheximide (CHX)-chase assay indicates the rapid degradation of mSOD1 at mitochondria by MITOL overexpression. Neuro2a cells transfected with indicated vectors were treated with CHX (10 μg/ml) for indicated times, and each lysate of whole, mitochondrial, or cytosolic fractions was immunoblotted with indicated antibodies. The relative protein levels of mSOD1 were quantified by densitometry (bottom panels). Solid and broken lines indicate the cotransfection with mock or MITOL, respectively. Error bars SD (n = 3). (C) Degradation of mSOD1 through the ubiquitin-proteasome system. Neuro2a cells transfected with indicated vectors were incubated with CHX (10 μg/ml) with or without MG132 (50 μM) for 3 h. The relative protein levels of mSOD1 were quantified by densitometry. Error bars, SD (n = 3). * p < 0.05; Student's t test. (D) mSOD1-dependent ROS generation was reduced by MITOL WT, but was enhanced by CS mutant. Twenty-four hours after cotransfection of Neuro2a cells with indicated vectors plus pDsRed-Mito, cells were incubated in differentiation medium for 24 h. Intracellular ROS generation in DsRed-Mito–positive Neuro2a cells was measured by flowcytometric analysis using H₂DCFDA staining. These represented results were from one of three experiments that produced similar results.