MOLECULAR CHARACTERIZATION OF MITOFILIN (HMP), A MITOCHONDRIA-ASSOCIATED PROTEIN WITH PREDICTED COILED-COIL AND INTERMEMBRANE SPACE TARGETING DOMAINS

We have identified and characterized a human protein of the mitochondr ia which we call mitofilin, Using monoclonal and polyclonal antibodies , we have isolated cDNA clones and characterized mitofilin biochemical ly, It appears as a 90 and 91 kDa doublet in western blots and is tran slated from a single 2.7 kb mRNA, Antibodies raised against cellular a nd bacterially-expressed protein give identical cytoplasmic immunofluo rescence and immunoblot results, Mitofilin co-localizes with mitochond ria in immunofluorescence experiments and co-purifies with mitochondri a, Double label studies show co-localization only with mitochondria an d not with Golgi or endoplasmic reticulum, Co-localization with mitoch ondria is retained when actin or tubulin are de-polymerized, and mitof ilin is expressed in all human cell types tested, The cDNA encodes a p olypeptide with a central alpha-helical region with predicted coiled c oil domains flanked by globular amino and carboxy termini, Unlike coil ed coil motor proteins, mitofilin is resistant to detergent extraction . The presence of mitochondrial targeting and stop-transfer sequences, along with the accessibility of mitofilin to limited proteolysis sugg ests that it resides predominantly in the intermembrane space, consist ent with immune-electron micrographs which show mitofilin mainly at th e mitochondrial periphery. The cDNA sequence of mitofilin is identical to that recently reported by Icho et al, (1994; Gene 144, 301-306) fo r a mRNA preferentially expressed in heart muscle (HMP), consistent wi th the high levels of mitochondria in cardiac myocytes.