The general mitochondrial matrix processing protease from rat liver: structural characterization of the catalytic subunit.
A critical step in the import of nuclear-encoded precursor proteins into mitochondria involves proteolytic cleavage of their amino-terminal leader peptides by processing proteases found in the mitochondrial matrix. We report here the characterization of the general matrix processing protease from rat liver mitochondria. The final enzyme preparation consisted of two polypeptides, a catalytically active 55-kDa subunit and a 52-kDa one. To deduce the complete primary structure of the 55-kDa subunit, we first sequenced its mature amino terminus and several tryptic peptides derived from the pure protein. Next, using mixed oligonucleotide primers that had sequences based on two of these peptides, we synthesized a partial cDNA probe by selective amplification of liver RNA with the polymerase chain reaction. The amplified probe was then used to obtain a nearly full-length clone from a rat liver cDNA library. This cDNA codes for 508 amino acid residues, including 16 residues of an amino-terminal leader peptide, the cleavage site of which is located two polypeptide bonds downstream from an arginine residue. The mature portion has a predicted molecular mass of 55.2 kDa; it shows 36% identity with the mitochondrial processing peptidases of Saccharomyces cerevisiae and Neurospora crassa. A conserved structural feature is a putative, negatively charged alpha-helix, located in the amino-terminal half of the subunit; this element might be important for the recognition of positively charged leader peptides characteristic of mitochondrial precursor proteins.
Kleiber
J
J
Yale University School of Medicine, Department of Human Genetics, New Haven, CT 06510.
Kalousek
F
F
Swaroop
M
M
Rosenberg
L E
LE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D046911:Macromolecular Substances; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
D046911:Macromolecular Substances; D004247:DNA; D010447:Peptide Hydrolases; D014357:Trypsin