Furthermore quantitative analysis of localization of CLIC CL

Furthermore, quantitative analysis of localization of CLIC1, CLIC4, and CLIC5 to the mitochondria in isolated adult cardiomyocytes (Fig. 3) revealed similar distribution of CLICs as observed in neonatal cardiomyocytes (Fig. 2). However, CLIC4 and CLIC5 showed a higher degree of colocalization to the mitochondria of adult cardiomyocytes in comparison to CLIC1 [Fig. 3 (I, III) (46.3±2.5%); 57.1±2.2%); vs. (35±4.1%), n=5, respectively]. There was no localization of CLIC1, CLIC4 or CLIC5 observed in the nucleus of adult cardiomyocytes (Fig 3D′, H′ and L′). D. melanogaster possesses a single CLIC ortholog, DmCLIC, and its localization in cardiac tubes was also tested. Cardiac tubes of flies showed localization of DmCLIC in mitochondria colabeled with anti-ATP synthase LY2584702 [Fig. 3(II, III)]. Clic, a protein-null mutant showed no significant labeling with anti-DmCLIC antibodies (Supplementary Fig. 3). Taken together, we have shown that mammalian cardiac CLIC4 and CLIC5 as well as DmCLIC localize to the mitochondria. Majority of mitochondrial proteins are known to possess canonical mitochondrial targeting sequence (MTS) (Omura, 1998), hence we investigated the presence of potential MTS in CLICs. Surprisingly, we did not detect any canonical MTS, and further in silico analysis (Ferre et al., 2013) revealed a very low probability (<0.005) for each CLIC protein being in mitochondria (Table 1). VDAC2 was taken as a control to validate the in silico analysis as it is a well-established OMM protein. All of our analysis algorithms predicted VDAC2 to be a mitochondrial protein. We further verified the presence of CLIC1, CLIC4 and CLIC5 in purified mitochondria following protocols described earlier (Singh et al., 2012). As shown in Fig. 4, CLIC4 and CLIC5 were present in the ultra-pure mitochondrial fraction (M3). All the three fractions obtained from 30% (v/v) Percoll gradient along with the crude mitochondria were analyzed for the presence of all other organelle markers. Percoll-purified mitochondrial M3 fraction was devoid of other organelles such as plasma membrane (pan-cadherin), nucleus (laminin b1), endoplasmic reticulum (GRP78 BiP), and Golgi apparatus (GM130), but showed the presence of mitochondrial markers (VDAC1 and Cox2) (Fig. 4). Although M1 and M2 fractions showed the presence of lamin B1 and pan cadherin, the ultrapure M3 fraction was devoid of any other cellular organelle contamination. Further in immuno organelle chemistry, CLIC4 and CLIC5 showed high degree of colocalization to ultra-pure mitochondrial fraction loaded with mitotracker (48.85±6.9% and 69.13±12.2%, respectively, n=3) but not CLIC1 (29.5±11.2%, n=3) (Fig. 5). These results in tandem with biochemical analysis strongly support that mammalian CLIC5 and CLIC4 are cardiac mitochondrial proteins. To further understand the localization and geographical distribution of CLIC4 and CLIC5 in the cardiac mitochondria, the Percoll-purified cardiac mitochondria were further separated into IMM and OMM fractions. As shown in the Western blot (Fig. 6A), CLIC4 showed higher levels of expression in OMM like VDAC1, and CLIC5-specific signals at ~30kDa and ~50kDa were enriched more in the IMM. To further analyze the distribution of CLIC4 and CLIC5, isolated mitochondria were imaged with the custom-built STED nanomicroscope. Confocal images were acquired before obtaining the STED images. As shown in Fig. 6II A–C and F–H, CLIC4 and CLIC5 showed differential distribution in isolated mitochondrion. CLIC4 had unique cluster distribution as reported for VDAC1 in isolated mitochondrion (Singh et al., 2012) but CLIC5 followed a continuous distribution.