Figure 2.

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.