Mutational analysis of both subunits from rat mitochondrial processing peptidase.
Rat liver mitochondrial processing peptidase (MPP) is the primary peptidase that cleaves leader peptides from nuclearly encoded mitochondrial proteins following their transport from the cytosol to the mitochondrial matrix. This enzyme consists of two nonidentical subunits that have overall similarity to each other and share certain amino acid motifs. These include the putative metal-ion binding HFLEH motif in the beta-subunit and the HFLEK motif of the alpha-subunit, as well as a possibly helical amino acid stretch bearing a high concentration of negatively charged residues about 70 amino acids downstream of these motifs in both subunits. In order to achieve a better understanding of the role of certain amino acids in rat MPP, we performed site-directed mutagenesis on both of its subunits. Our results show that whereas both histidines and the glutamate of the HFLEH motif in the beta-subunit are crucial for MPP function, this holds true only for the glutamate in the related HFLEK motif in the alpha-subunit. In addition, functionally important negatively charged residues in the region 70 amino acids downstream occur only in the beta-subunit and not in the alpha-subunit. This indicates a functional asymmetry between the subunits, with the beta-subunit containing a majority of residues participating in the active center.
Department of Genetics, Yale School of Medicine, New Haven, Connecticut 06520-8005, USA.
H M
HM
Striebel
P
P
Rysavy
J
J
Adamec
J
J
Spizek
F
F
Kalousek
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000096122:Mitochondrial Processing Peptidase
D046911:Macromolecular Substances; D008666:Metalloendopeptidases; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000096122:Mitochondrial Processing Peptidase