Purification and characterization of a processing protease from rat liver mitochondria.

Ou, Weijia; Ito, Akio; Okazaki, Hiroshi; Omura, Tsuneo

doi:10.1002/j.1460-2075.1989.tb08400.x

Cited by 129 publications

(60 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1A, lane 4) migrated at the same position of pMe 2 GlyDH: it is named The boiling of detergent-solubilised mitoplasts, prior to the addition to the £avinylation assay, resulted in a complete loss of £avinylated proteins, con¢rming that a mitochondrial protein component(s) is involved in £avinylation of Me 2 GlyDH. The treatment of solubilised mitoplasts with EDTA (1 mM) to inhibit MPP [13] left the RL translated precursor completely unprocessed, but still e⁄ciently £avinylated; in fact, about 50% of the total amount of RL translated protein was present as p FAD . Thus, precursor processing is not strictly required to allow the £avinylation to proceed.…”

Section: Resultsmentioning

confidence: 99%

Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

2002

View full text Add to dashboard Cite

The £avinylation and the presequence processing of the mitochondrial matrix enzyme dimethylglycine dehydrogenase (Me 2 GlyDH) were investigated with the reticulocyte lysate translated precursor (pMe 2 GlyDH) added to solubilised mitoplasts of rat liver mitochondria. The £avinylation of pMe 2 -GlyDH was strictly dependent on the addition of mitochondrial protein(s), among which the mitochondrial £avinylation stimulating factor [Brizio C., et al. (2000) Eur. J. Biochem 267, 4346^4354], that actively promotes holo-Me 2 GlyDH formation. The precursor processing, that accompanies the biogenesis of the enzyme, was not required to allow the £avinylation to proceed. The comparison of the time course of the £avinylation and the processing of pMe 2 GlyDH demonstrated that the covalent attachment of the £avin moiety preceded the presequence processing by mitochondrial processing peptidase. ß

show abstract

Section: Resultsmentioning

confidence: 99%

Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

2002

View full text Add to dashboard Cite

show abstract

“…Materials-MPP was purified from bovine or rat liver mitochondria according to the method of Ou et al (6). Solvents for peptide synthesis were purchased from Kanto Chemical Co. Ltd. (Tokyo).…”

Section: Methodsmentioning

confidence: 99%

Analysis of Elements in the Substrate Required for Processing by Mitochondrial Processing Peptidase

Ogishima

Niidome²,

Shimokata

et al. 1995

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have recently demonstrated that synthetic peptides modeled on the extension peptide of malate dehydrogenase can be a good substrate of mitochondrial processing peptidase and that arginine residues present at positions ؊2 or ؊3 and distant from the cleavage point were important for recognition by the enzyme (Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719 -24722). We further investigated the elements required for substrates of the protease. To analyze the reaction by a more rapid yet quantitative method, we have developed intramolecularly quenched fluorescent substrates. Using the fluorogenic substrates we demonstrated that at least one of the proline and glycine between the distal and proximal arginine residues was also important while other connecting sequences were dispensable. In addition, the protease showed considerable preference for aromatic and, to a lesser extent, hydrophobic amino acids in the P 1 -position. These results together with the previous data suggest that the proximal and distal arginine residues, proline and/or glycine between them, and P 1 amino acid could be critical determinants for the specific cleavage of the substrates by the protease.Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and transported into their correct mitochondrial component. The majority of them carry N-terminal extension peptides that target the protein molecules to the organelle. Mitochondrial processing peptidase (MPP) 1 is localized in the mitochondrial matrix and is responsible for proteolytic cleavage of the extension peptides after or during the transportation (1-3). The enzyme is a metalloprotease and forms a heterodimer consisting of structurally related ␣-and ␤-subunits (4 -11). We have recently demonstrated that the ␤-subunit is a catalytic one (12). MPP acts exclusively on the precursor forms of mitochondrial proteins, whose extension peptides are heterogeneous in sequence (13-15). The absence of apparent sequence homologies raises a question about how MPP specifically recognizes the extension peptides and cleaves them at a single site. Attempts have been made to solve the question, mainly by use of in vitro translated and radiolabeled mutants of mitochondrial precursors. By detecting mature forms from the in vitro translated precursors on SDS-polyacrylamide gel electrophoresis followed by fluorography, the processing activity has so far been evaluated. Those studies pointed out the importance of basic amino acids near the C terminus and neutral amino acids in the middle portion of the extension peptides (16 -20). The method, however, is time-consuming, and the obtained data are not quantitative. Thus the conventional method is unsuitable for kinetic analysis of the reaction. To overcome the limitation of the assay method, we developed a new method (21), which employed as the substrate synthetic peptides that were modeled on the extension peptide of rat malate dehydrogenase. This method enabled us to reveal the importance of argini...

show abstract

“…MPP alone has a very low processing activity (111). Subsequently, processing peptidase was purified from yeast mitochondria (112) and rat liver mitochondria (113). Neurospora crassa, MPP and PEP are recovered as individual proteins during isolation; in yeast and more so in rat liver, the two components form a complex.…”

Section: Processing Enzymesmentioning

confidence: 99%

The Mitochondrial Protein Import Apparatus

Pfanner¹

1990

Annual Review of Biochemistry

View full text Add to dashboard Cite

Purification and characterization of a processing protease from rat liver mitochondria.

Cited by 129 publications

References 63 publications

Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

Analysis of Elements in the Substrate Required for Processing by Mitochondrial Processing Peptidase

The Mitochondrial Protein Import Apparatus

Contact Info

Product

Resources

About