ausblenden:
Schlagwörter:
ATP-Dependent Proteases
Adenosine Triphosphate/*metabolism
Amino Acid Sequence
Catalysis
Chromatography, Affinity
Chromatography, Gel
Cloning, Molecular
DNA Primers
Enzyme Precursors/chemistry/*metabolism
Mitochondria/*enzymology
Mitochondrial Proteins
Molecular Sequence Data
Mutagenesis, Site-Directed
Polymerase Chain Reaction
*Protein Processing, Post-Translational
Recombinant Proteins/chemistry/isolation & purification/metabolism
Saccharomyces cerevisiae/*enzymology
*Saccharomyces cerevisiae Proteins
Serine Endopeptidases/chemistry/isolation & purification/*metabolism
Zusammenfassung:
The biogenesis of the ATP-dependent PIM1 protease of mitochondria was studied by mutational analysis. The ATPase and proteolytic activities of PIM1 were shown to be essential for mitochondrial function. A proteolytically inactive mutant form of PIM1 protease accumulated as a pro-form in mitochondria, revealing a two-step processing of PIM1: the matrix targeting signal is removed by the mitochondrial processing peptidase and then a pro-region of 61 amino acids is cleaved off in an autocatalytic reaction. This latter process depended on the ATP-dependent assembly of PIM1 protease subunits and can occur by an intermolecular and, most probably, also an intramolecular pathway. The respiratory competence of cells harboring mutant PIM1 protease lacking the pro-region was strongly impaired. Subcellular fractionation revealed a cytosolic localization of mutant PIM1 protease. This demonstrates the requirement for the propeptide for efficient sorting of PIM1 protease to mitochondria.
