Lon1 Protease Controls PentatricoPeptide-Repeat (PPR)-Mediated RNA Processing in Arabidopsis Mitochondria.

Selective proteolysis maintains cellular homeostasis by preventing the accumulation of unfolded or misfolded proteins, which can form deleterious aggregates. Lon is a highly conserved AAA+ (ATPases Associated with diverse cellular Activities) protease in bacteria and eukaryotic organelles, essential for protein quality control. However, the mechanisms of Lon-mediated protein substrate recognition and degradation remain unknown in plant mitochondria. Here, we identify mitochondrial substrates of Arabidopsis Lon1 protease by using a proteolytically inactive Lon1-trap variant in a lon1 mutant background, which exhibits growth retardation due to mitochondrial dysfunction. The identified substrates participate in key mitochondrial pathways, including energy metabolism, transcription and translation, highlighting Lon1's role in maintaining mitochondrial integrity. Notably, we uncover a set of bona fide Lon1 targets, including enzymes involved in calcium transport, lipoic acid synthesis, heat shock responses and PentatricoPeptide-Repeat (PPR) proteins, which are key regulators of mitochondrial gene expression. Strikingly, PPR proteins are highly overrepresented in the lon1 proteome, correlating with defects in group II intron splicing and RNA editing, particularly in transcripts encoding Complex I subunits, cytochrome c biogenesis factors, Complex IV components and ribosomal proteins. Our findings reveal a novel regulatory role for Lon1 in mitochondrial RNA processing and maturation through selective proteolysis of PPR proteins.