Catalysis, subcellular localization, expression and evolution of the targeting peptides degrading protease, AtPreP2

Area of Science:

• Plant molecular biology
• Protein biochemistry
• Organelle biology

Background:

• Presequence protease (PreP) degrades mitochondrial and chloroplast targeting peptides.
• Arabidopsis thaliana has two PreP homologs: AtPreP1 and AtPreP2.
• AtPreP1 is dual-targeted and degrades both mitochondrial and chloroplast targeting peptides.

Purpose of the Study:

• To characterize the proteolytic and targeting properties of AtPreP2.
• To compare AtPreP2 with AtPreP1.
• To investigate the in vivo targeting of AtPreP2.

Main Methods:

• Overexpression and purification of AtPreP2.
• In vitro import assays using isolated organelles.
• Transient expression in tobacco protoplasts and leaves.
• Shotgun mass spectrometry of purified chloroplasts.
• RT-PCR analysis for gene expression.

Main Results:

• AtPreP2 exhibits distinct proteolytic subsite specificity compared to AtPreP1.
• AtPreP2 precursor is dual-targeted to mitochondria and chloroplasts in vitro.
• AtPreP2 targeting peptide directs GFP to both organelles in vivo.
• AtPreP1 and AtPreP2 are confirmed to be chloroplast-targeted in vivo.
• AtPreP1 and AtPreP2 show differential expression patterns.

Conclusions:

• AtPreP2 is a dual-targeted protease with unique proteolytic activity.
• The two Arabidopsis PreP homologs may have diverged through subfunctionalization.
• Understanding PreP function is crucial for plant organelle biogenesis.