Molecular cloning and characterization of a mouse homolog of bacterial ClpX, a novel mammalian class II member of the Hsp100/Clp chaperone family

In this paper, we present the molecular cloning and characterization of a m urine homolog of the Escherichia coli chaperone ClpX, Murine ClpX shares 38 % amino acid sequence identity with the E, coli homolog and is a novel memb er of the Hsp100/Clp family of molecular chaperones. ClpX localizes to huma n chromosome 15q22.2-22.3 and in mouse is expressed tissue-specifically as one transcript of similar to 2.9 kilobases (kb) predominantly within the li ver and as two isoforms of similar to 2.6 and similar to 2.9 kb within the testes. Purified recombinant ClpX displays intrinsic ATPase activity, with a K-m of similar to 25 mu M and a V-max of similar to 660 pmol min(-1) mu g (-1), which is active over a broad range of pH, temperature, ethanol, and s alt parameters. Substitution of lysine 300 with alanine in the ATPase domai n P-loop abolishes both ATP hydrolysis and binding. Recombinant ClpX can al so interact with its putative partner protease subunit ClpP in overexpressi on experiments in 293T cells. Subcellular studies by confocal laser scannin g microscopy localized murine ClpX green fluorescent protein fusions to the mitochondria. Deletion of the N-terminal mitochondrial targeting sequence abolished mitochondrial compartmentalization, Our results thus suggest that murine ClpX acts as a tissue-specific mammalian mitochondrial chaperone th at may play a role in mitochondrial protein homeostasis.