Mitochondrial outer membrane proteins were removed from mitochondria by nonionic detergent digitonin treatment. or GST-PHD CS mutant (Figure 1B). This indicated Rabbit polyclonal to PPP1R10 that PHD in MITOL has an E3 ubiquitin ligase activity. Northern blot analysis showed the ubiquitous expression of MITOL in various human tissues (Figure 1C). To ascertain expression of MITOL a specific anti-MITOL antibody was raised against a peptide corresponding to amino acids 257C279 of MITOL. Western blot analysis with anti-MITOL antibody demonstrated the expression of MITOL as an 30 kDa band in both COS-7 and HeLa cells, but not with control, preimmune serum (Figure 1D). In addition, an enhanced MITOL signal in MITOL expression vector-transfected cells verified the specificity of this antibody and the position of MITOL Isochlorogenic acid A protein band. Open in a separate window Figure 1 Identification of Isochlorogenic acid A MITOL, a novel membrane-bound E3 ubiquitin ligase. (A) Amino-acid sequence of human MITOL. PHD motif and predicted four-transmembrane domains are shown by an underline and shadow lines, respectively. Anti-MITOL antibody was raised against the peptide indicated by dots above the amino acids. (B) E3 ubiquitin ligase activity in GST-fused MITOL-PHD, but not CS mutant (C65S, C68S). ubiquitin ligase assay using purified GST, GST-PHD and GST-PHD CS mutants was performed as described in Materials and methods. (C) Northern blot analysis of MITOL in various human tissues. A full-length human MITOL cDNA was used as a probe. (D) Expression of MITOL protein in COS-7 and HeLa cell lines. Lysates of COS-7, HeLa and MITOL expression vector-transfected HeLa cells were immunoblotted with control preimmune or immune serum containing anti-MITOL antibody. In order to examine subcellular distribution of MITOL in HeLa cells, immunohistochemical analysis using anti-MITOL antibody was performed and is shown in Figure 2A (upper panels). Endogenous MITOL was colocalized with mitochondrial marker MitoTracker, suggesting that MITOL is a mitochondrial protein. Likewise, HeLa cells expressing Myc-tagged MITOL were immunostained with anti-Myc antibody and MitoTracker. Myc-tagged MITOL was also colocalized with mitochondria (Figure 2A, lower panels). To confirm the mitochondrial localization of MITOL, cytosolic and mitochondrial fractions of normal HeLa or HeLa cells expressing Myc-tagged MITOL were isolated and MITOL localization was determined by immunoblotting using anti-MITOL (Figure 2B, left) Isochlorogenic acid A or anti-Myc antibodies (Figure 2B, right). As expected, MITOL was localized in the mitochondrial fraction. As the mitochondrial fraction is known to contain a part of endoplasmic reticulum (ER) proteins, mitochondrial and ER-rich fractions were separated and MITOL localization was examined using anti-PDI (ER marker) and anti-Tom20 antibodies (mitochondria marker). As shown in Figure 2C, MITOL was not detected in the ER-rich fraction, indicating the specific localization of MITOL in mitochondria. Carbonate extraction assay showed that MITOL behaved like an integral membrane protein. This result was consistent with predicted four-transmembrane structure (Figure 2D). We next examined whether MITOL localized at the outer or inner membrane of mitochondria. Purified intact mitochondria prepared from HeLa cells were treated with trypsin, which attacked the cytosolic domain of the outer membrane proteins. Treatment with trypsin did not affect the inner membrane protein Tim23, whereas it degraded MITOL as well as the outer membrane protein Tom20 (Figure 2E). Mitochondrial outer membrane proteins were removed from mitochondria by nonionic detergent digitonin treatment. The digitonin solubility of MITOL was consistent with that of Tom20 but not Tim23 (Figure 2E). To determine the topology of the N-terminus, trypsin protection assay on mitochondria isolated from cells expressing an N-terminally HA-tagged MITOL was performed. As shown in Figure.
