A HUMAN HOMOLOG OF ESCHERICHIA-COLI CLPP CASEINOLYTIC PROTEASE - RECOMBINANT EXPRESSION, INTRACELLULAR PROCESSING AND SUBCELLULAR-LOCALIZATION

We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease [Bross , Andresen, Knudsen, Kruse and Gregersen (1995) FEES Lett. 377, 249-25 2]. In the present study, synthesis, intracellular processing and subc ellular localization of hClpP have been analysed in intact cells and i n a cell-free system, Using pulse-labelling/immunoprecipitation of Cha ng cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx. 39 and 37 kDa. A pulse-chase experiment showed that these bands were converted into one mature-enz yme band with a molecular mass of approx. 32 kDa that was stable for a t least 24 h. The 37 kDa band comigrated with a band produced upon exp ression of full-length hClpP in E. coli, and the 32 kDa band co-migrat ed with the product of E. coil-expressed hClpP in which the 56 N-termi nal residues had been deleted, indicating that the 37 kDa moiety repre sents the precursor and that approx. 56 residues are cleaved off durin g maturation. The processing of hClpP in intact cells was dependent on mitochondrial membrane potential. These results were confirmed in an import assay system using in vitro transcription and translation direc ted by the hClpP cDNA and isolated rat liver mitochondria. No protease activity towards a series of fluorogenic peptides could be observed i n extracts of Chang cells overexpressing hClpP, indicating that the pr otease may not be active without co-factors. Immunofluorescence studie s using confocal-laser-scanning microscopy showed colocalization of th e hClpP and the mitochondrially located Hsp60 (heat-shock protein 60). Taken together, the results reported here show that hClpP is localize d inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochon drial proteins.