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Evidences for adenine nucleotide binding in the subunits of Neurospora mitochondrial processing peptidase
R Botweva1, B Fillippi, B Salvato
1Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Abstract:
The mitochondrial processing peptidase (MPP) of Neurospora crassa consists of two subunits termed alpha-and beta-MPP. Here we present spectroscopic and chromatographic studies indicative of adenine nucleotide binding in the two MPP subunits. ADP was identified as the cofactor of alpha-MPP and ATP as the cofactor of beta-MPP. The nucleotides are not covalently bound and exert strong control on the conformational and functional properties of the subunits. The ADP cofactor of alpha-MPP seems to be of utmost importance for the proteolytic activity because no processing of the precursor protein was registered in the assay containing alpha-MPP depleted of ADP and native beta-MPP. On the contrary, with native alpha-MPP and depleted of ATP beta-MPP almost 100% processing activity could be measured. Very strong increase of the intensity and significant changes in the shape and maximum position of the protein fluorescence spectra were observed after removal of the adenine cofactors of alpha- and of beta-MPP.
Insights
Neurospora crassa mitochondrial processing peptidase (MPP) subunits bind adenine nucleotides. ADP is crucial for alpha-MPP activity, while ATP binds beta-MPP, influencing protein conformation and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The mitochondrial processing peptidase (MPP) is essential for protein maturation in mitochondria.
- MPP from Neurospora crassa comprises two subunits: alpha-MPP and beta-MPP.
Purpose of the Study:
- To investigate adenine nucleotide binding to Neurospora crassa MPP subunits.
- To determine the role of these nucleotide cofactors in MPP function and conformation.
Main Methods:
- Spectroscopic studies to detect nucleotide binding.
- Chromatographic techniques for cofactor identification and removal.
- Enzyme activity assays to assess proteolytic processing.
Main Results:
- Adenine nucleotides were identified as cofactors for both alpha-MPP (ADP) and beta-MPP (ATP).
- Nucleotide binding significantly impacts the conformational and functional properties of MPP subunits.
- ADP is critical for alpha-MPP's proteolytic activity; its removal abolishes processing.
- Removal of ATP from beta-MPP had a lesser impact on overall processing activity.
Conclusions:
- Adenine nucleotides are non-covalently bound cofactors regulating Neurospora crassa MPP activity.
- ADP binding to alpha-MPP is essential for its proteolytic function.
- The binding and removal of these cofactors induce significant changes in protein fluorescence spectra.