Single amino acid mutations in the S. cerevisiae rhomboid peptidase, Pcp1p, alter mitochondrial morphology.

Key to mitochondrial activities is the maintenance of mitochondrial morphology, specifically cristae structures formed by the invagination of the inner membrane that are enriched in proteins of the electron transport chain. In S. cerevisiae, these cristae folds are a result of the membrane fusion activities of Mgm1p and the membrane bending properties of ATP synthase oligomerization. An additional protein linked to mitochondrial morphology is Pcp1p, a serine protease responsible for the proteolytic processing of Mgm1p. Here we have used hydroxylamine-based random mutagenesis to identify amino acids important for Pcp1p peptidase activity. Using this approach we have isolated five single amino acid mutants that exhibit respiratory growth defects that correlate with loss of mitochondrial genome stability. Reduced Pcp1p protease activity was confirmed by immunoblotting with the accumulation of improperly processed Mgm1p. Ultra-structural analysis of mitochondrial morphology in these mutants found a varying degree of defects in cristae organization. However, not all of the mutants presented with decreased ATP synthase complex assembly as determined by BN-PAGE. Together, these data suggest that there is a threshold level of processed Mgm1p required to maintain ATP synthase super complex assembly and mitochondrial cristae organization. This article is protected by copyright. All rights reserved.