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Characterization of the mitochondrial processing peptidase of Neurospora crassa
M Arretz1, H Schneider, B Guiard
1Institut für Physiologische Chemie, Universität München, Federal Republic of Germany.
Abstract:
The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH-terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping.
Insights
The mitochondrial processing peptidase (MPP) requires both alpha and beta subunits for function. This enzyme recognizes specific C-terminal sequences in preproteins for cleavage, distinct from import signals.
Area of Science:
- Mitochondrial protein import
- Protease function
- Neurospora crassa biochemistry
Background:
- Mitochondrial processing peptidase (MPP) is crucial for protein maturation.
- MPP in Neurospora crassa consists of alpha and beta subunits.
- Understanding MPP substrate recognition is key to protein import.
Purpose of the Study:
- To characterize the Neurospora crassa MPP.
- To identify the structural elements recognized by MPP on its substrates.
- To differentiate MPP recognition from mitochondrial import machinery recognition.
Main Methods:
- Purification of alpha-MPP from E. coli and beta-MPP from mitochondria.
- In vitro processing assays using a fusion protein (precytochrome b2-DHFR) as substrate.
- Competition assays with presequence peptides.
- Site-directed mutagenesis of the precytochrome b2 targeting sequence.
Main Results:
- Both alpha- and beta-MPP subunits are essential for processing.
- MPP cleaves the cytochrome b2 matrix targeting signal, producing a 31-amino acid presequence.
- The presequence peptide is a weak competitive inhibitor, indicating lower affinity.
- MPP primarily recognizes COOH-terminal regions of the presequence, not the N-terminal amphipathic helix.
- Mutations at positions -2 and +1 relative to the cleavage site affect processing but not import.
- Non-cleavable preproteins are still recognized by MPP.
Conclusions:
- Mitochondrial processing peptidase requires both subunits for activity.
- MPP substrate recognition focuses on the COOH-terminal part of the presequence.
- Distinct structural elements are recognized by MPP and the import machinery, though they may overlap.