OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

Area of Science:

• Mitochondrial biology
• Protease biochemistry
• Cellular dynamics

Background:

• Mitochondrial morphology is crucial for cellular function and is regulated by the proteolytic processing of OPA1, a key inner membrane GTPase.
• The mitochondrial rhomboid protease PARL and the m-AAA protease paraplegin were previously implicated in OPA1 cleavage.

Purpose of the Study:

• To identify the specific proteases responsible for OPA1 cleavage and characterize their substrate specificities.
• To investigate the evolutionary conservation and divergence of proteases involved in mitochondrial dynamin-like GTPase processing.

Main Methods:

• Mass spectrometry to characterize OPA1 isoforms.
• Reconstitution of OPA1 processing in yeast using homologous and heterologous proteases.
• Analysis of OPA1 processing in yeast and mammalian cell lines with genetic deletions or varying protease subunit compositions.

Main Results:

• Yeast rhomboid proteases (PARL, Pcp1) did not cleave OPA1, although they processed the yeast homologue Mgm1.
• Formation of small OPA1 isoforms was dependent on the m-AAA protease subunits Yta10 and Yta12 in yeast.
• Mammalian m-AAA proteases, particularly homo-oligomeric complexes of Afg3l1/Afg3l2 or AFG3L2, showed higher OPA1 cleavage efficiency than paraplegin-containing complexes.
• OPA1 processing occurred normally in mammalian cells lacking paraplegin or PARL, suggesting functional redundancy or alternative pathways.

Conclusions:

• The m-AAA protease, not rhomboid proteases, is the primary enzyme responsible for OPA1 processing in mammalian mitochondria.
• The subunit composition of m-AAA proteases dictates their substrate specificity for OPA1.
• A significant evolutionary divergence in the proteases mediating mitochondrial dynamin-like GTPase processing has occurred between yeast and mammals.