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 murine homolog of the Escherichia coli chaperone ClpX. Murine ClpX shares 38% amino acid sequence identity with the E. coli homolog and is a novel member of the Hsp100/Clp family of molecular chaperones. ClpX localizes to human chromosome 15q22.2-22.3 and in mouse is expressed tissue-specifically as one transcript of approximately 2.9 kilobases (kb) predominantly within the liver and as two isoforms of approximately 2.6 and approximately 2.9 kb within the testes. Purified recombinant ClpX displays intrinsic ATPase activity, with a Km of approximately 25 microM and a Vmax of approximately 660 pmol min-1 microgram-1, which is active over a broad range of pH, temperature, ethanol, and salt parameters. Substitution of lysine 300 with alanine in the ATPase domain P-loop abolishes both ATP hydrolysis and binding. Recombinant ClpX can also interact with its putative partner protease subunit ClpP in overexpression experiments in 293T cells. Subcellular studies by confocal laser scanning 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 that may play a role in mitochondrial protein homeostasis.
Santagata
S
S
Mount Sinai School of Medicine, New York, New York 10029, USA. santas01@doc.mssm.edu
Bhattacharyya
D
D
Wang
F H
FH
Singha
N
N
Hodtsev
A
A
Spanopoulou
E
E
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002884:Chromosomes, Human, Pair 15; D003001:Cloning, Molecular; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism; D013737:Testis / Q000737:chemistry; D014162:Transfection
D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities