File S1 - NOA1, a Novel ClpXP Substrate, Takes an Unexpected Nuclear Detour Prior to Mitochondrial Import

Supporting table and figures. Table S1, Screening of nuclear compounds initiating nuclear accumulation of NOA1. A panel of 1280 compounds (LOPAC1280 panel, Sigma Aldrich) was screened for factors causing accumulation of NOA1 in nuclei of C2C12 cells. The primary screen for compounds was evaluated by analysis of nuclear fluorescence caused by transfected NOA1-EGFP six hours after treatment with 100 µM concentrated substance in DMSO. 3.2% (41/1280) of the compounds induced increased nuclear accumulation of NOA1. 30.08% (385/1280) of the compounds induced cell death at a concentration of 100 µM. Substances causing severe toxic effects on C2C12 cells were excluded. Effects of compounds were compared to negative controls (1% DMSO and water), which did have no effects on the subcellular localization of NOA1-EGFP. All 41 compounds that were testing positive were subjected to a secondary screen based on the accumulation of endogenous NOA1 in nuclei of C2C12 cells. 17 out of 41 (1.33% of total) retested compounds consistently initiated accumulation of endogenous NOA1 in the nucleus of more than 10% of the cells. Four substances (trequinsine, quazinone, flunarizine and amiloride) were further analyzed. Flunarizine yielded the strongest effects and was therefore employed for subsequent studies. Figure S1, Rabbit polyclonal anti-NOA1 antibody is specific for precursor and mature NOA1 protein. To assess specificity of the rabbit polyclonal anti-NOA1 antibody used in this study we expressed C-terminally Flag tagged NOA1 in C2C12 myoblasts and analyzed the protein lysates by Western blotting. Flag antibody detected the larger precursor and the shorter, mature, N-terminally processed NOA1 protein. Similar result was obtained with the antibody raised against NOA1. Although highly specific, the endogenous NOA1 protein expression was below threshold for detection by Western blot. Figure S2, Flunarizine induces accumulation of NOA1 in the nucleus. (A) Immunofluorescence staining of NOA1-transfected C2C12 cells. NOA1 mainly localizes in mitochondria as demonstrated by co-localization with the mitochondrial marker protein Tom20. (B) Six hour treatment with flunarizine (100 µM), identified by screening a LOPAC library for compounds altering subcellular localization of NOA1, leads to rapid accumulation of NOA1-EGFP in the nucleus of C2C12 cells. (C) FACS analysis of non-transfected C2C12 cells after Flunarizine treatment (100 µM for 6 hours) shows no increase of apoptosis. (D) Inhibition of translation by 10 ng/ml Cycloheximide (CHX) does not affect mitochondrial localization of NOA1-EGFP. (E) Combined treatment with Flunarizine (100 µM) and CHX (10 ng/ml) prevents accumulation of NOA1 in the nucleus indicating that only newly translated NOA1 is targeted to the nucleus. (F) Combined CHX and flunarizine treatment of NOA1-EGFP transfected C2C12 cells reduces the percentage of NOA1-EGFP positive non-viable cells compared to flunarizine treatment alone. Cell viability was assessed by counting the number of attached and non-rounded EGFP-positive cells. Scale bars: 10 µm. (PDF)