Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase

Area of Science:

• Mitochondrial protein import and processing
• Molecular biology of ribosomal proteins
• Plant biochemistry

Background:

• Nuclear-encoded mitochondrial proteins are synthesized in the cytosol and imported into mitochondria.
• Rice mitochondrial ribosomal protein S14 (RPS14) is initially synthesized as a polyprotein precursor, preSDHB-RPS14.
• Mitochondrial processing peptidase (MPP) is crucial for processing various mitochondrial precursors.

Purpose of the Study:

• To investigate the role of mitochondrial processing peptidase (MPP) in processing the rice RPS14 polyprotein precursor.
• To determine the cleavage sites and mechanism by which MPP processes the preSDHB-RPS14 polyprotein.
• To understand the substrate recognition and processing preferences of MPP for this specific polyprotein.

Main Methods:

• In vitro cleavage assays using purified yeast MPP and the preSDHB-RPS14 polyprotein.
• Determination of cleavage sites within the polyprotein precursor.
• Mutational analysis of the connector region between SDHB and RPS14 domains.
• Assessment of MPP's preference for unfolded versus folded protein structures.

Main Results:

• Purified yeast MPP efficiently cleaved both the N-terminal presequence and the connector region between SDHB and RPS14 in the rice precursor.
• Cleavage within the connector region occurred more rapidly than cleavage of the N-terminal presequence.
• The cleavage site in the connector region contained an MPP processing motif, similar to the N-terminal presequence.
• MPP demonstrated a preference for the unfolded conformation of the preSDHB-RPS14 polyprotein.

Conclusions:

• The general mitochondrial processing peptidase (MPP) is essential for processing the rice preSDHB-RPS14 polyprotein.
• MPP can cleave the internal connector region between SDHB and RPS14, in addition to the N-terminal presequence.
• MPP's recognition of the unfolded polyprotein suggests it may cleave the precursor during translocation across the inner mitochondrial membrane.
• These findings provide insights into the complex processing events of mitochondrial polyproteins.